Glycerol, propoxylated, esters with acrylic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Results of studies with reliabilities 1 and 2:

Gene mutation in bacteria:
-S. typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA, with and without metabolic activation (Aroclor-induced), OECD 471: negative (BASF, 2004 )

-S. typhimurium TA98, TA100, TA1535, TA1537 and TA1538, with and without metabolic (Aroclor-induced), Ames et al.: negative (Cognis, 1982)
-S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100, with and without metabolic activation (Aroclor-induced) , OECD 471: negative (Cytec, 1983)
-S. typhimurium TA 1538, TA 1535, TA 1537, TA 98 and TA 100, with and without metabolic activation (Aroclor-induced) Ames et al.: negative (Cytec, 1984)

Cytogenicity in mammalian cell:
-Chromosome Aberration, human lymphocytes, with and without metabolic activation (Aroclor-induced) OECD 473: negative (Cytec, 1983)

Gene mutation in mammalian cells:

-HPRT, V79, with and without metabolic activation ( Phenobarbital/ β-naphthoflavone induced) OECD guideline 476: negative (ReachCentrum, 2020)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

19 May 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Batch No.: GK0561/158
Storage: Room temperature
Stability: guaranteed until June 8, 2005

Target gene:

his operon

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:

Type and composition of metabolic activation system:
- source of S9 : Aroclor 1254 induced rat liver
- method of preparation of S9 mix: a sufficient amount of S-9 fraction was thawed at room temperature and 1 volume of S-9 fraction is mixed with 9 volumes of S-9 supplement (cofactors). The concentrations of the cofactors in the S-9 mix are:
8 mM MgCl2
33 mM KCI
5 mM Glucose-6-phosphatase
4 mM NADP
15 mM Phosphate buffer (pH 7.4)
The phosphate buffer (6) is prepared by mixing an Na2HPO4 solution with an NaH2PO4 solution in a ratio of about 4 :1.
- S9 in the final culture medium : 0.5 mL
- quality controls of S9: To demonstrate the efficacy of the S-9 mix in this assay, the S-9 batch was characterized with benzo(a)pyrene

Test concentrations with justification for top dose:

20 µg - 5 000 µg/plate (Standard plate test)
20 µg - 5 000 µg/plate (Preincubation test)

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of vehicle:Due to the limited solubility of the test substance in water, DMSO was selected as the vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Untreated negative controls:

yes

Remarks:

Sterility and Vehicle controls

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene; N-methyl-N'-nitro-N-nitrosoguanidine; 4-nitro-o-phenylendiamine; 9-a minoacridine (AAC); 4-nitroquinoline-N-oxide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension (soft agar;amino acid solution;test solution or vehicle;fresh bacterial culture; S-9 mix or phosphate buffer were mixed and poured onto agar plates)

DURATION (Preincubation and Standardtest were performed)
- Preincubation period: 20 min
- Exposure duration: 48h-72h
- Fixation time (start of exposure up cell count): 48h-72h

SELECTION AGENT: histidine

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (number of revertants were counted)

Evaluation criteria:

The test chemical is considered positive in this assay if the following criteria are met:
- A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain.

A test substance is generally considered nonmutagenic in this test if:
-The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Toxicity detected by a
- decrease in the number of revertants
- clearing or diminution of the background lawn (= reduced his- or trp- background growth)
- reduction in the titer

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

slight decrease in the number of revertantswas observed depending on the strain and test conditions at doses ≥ 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

slight decrease in the number of revertantswas observed depending on the strain and test conditions at doses ≥ 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: No precipitation was found.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : Please refer to Table 1 to 10.

Ames test:
- Signs of toxicity : A slight decrease in the number of revertants was occasionally observed in the standard plate test depending on the strain and test conditions at 5 000 pg/plate. In the preincubation assay a weak bacteriotoxicity (slight decrease in the number of revertants and / or a slight reduction in the titer) was observed depending on the strain and test conditions at doses > 2500 pg/plate.
- Individual plate counts: Please refer to Table 1 to 10
- Mean number of revertant colonies per plate and standard deviation : Please refer to Table 1 to 10

HISTORICAL CONTROL DATA
- Positive historical control data: Please refer to Table 11 to 15
- Negative historical control data: Please refer to Table 16 to 20

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 06, 1983 to September 21, 1983

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: obtained from human blood

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium supplemented with 20% foetal calf serum (RPMI + FCS) reconstituted with phytohaemagglutinin
- Properly maintained: Yes

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: Aroclor 1254 induced rat liver
- Method of preparation of S9 mix:
S9 fraction: 0.1 mL
0.4 M MgCl2: 0.02 mL
0.2 M sodium orthophosphate buffer pB 7.4: 0.5 ml;
1 M glucose-6-phosphate: 0.005 mL
0.1 M NADP: 0.04 mL
distilled water: 0.335 mL
All the cofactors will be filter sterilized before use except for the S-9 fraction.
- Volume of S9 mix in the final culture medium: 0.25 mL
- Quality controls of S9: enzymatic activity (tested before use with the carcinogen 7,12-dimethylbenz(a)anthracene.)

Test concentrations with justification for top dose:

Preliminary toxicity test: 0.977, 1.953, 3.906, 7.813, 15.625, 31.25, 62.5, 125, 250 and 500 µg/mL
Main Test:
Without S-9 mix: 0.4, 2.0 and 4.0 µg/mL
With S-9 mix: 0.8, 4.0 and 8.0 µg/mL

Vehicle / solvent:

- Vehicle used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

mitomycin C

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1x10^6 cells
- Test substance added in medium: in suspension

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 48 hours
- Exposure duration/duration of treatment: 24 hours

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor: Colchicine 0.25 µg/mL for 2 hours
- Methods of slide preparation and staining technique used including the stain used:
After the incubation period, each 1 ml of cell suspension was transferred to a conical centrifuge tube, made hypotonie by addition of 4 ml distilled water, and incubated at room temperature for 10 minutes. The cultures were then centrifuged at 110 x 'g' for 10 minutes, the supernatant discarded and 4 ml of freshly prepared fixative (3 parts methanol : 1 part glacial acetic acid v/v) added. The cell pellets were left in fixative overnight. The pellets were then resuspended by repeated aspiration through a 20 gauge needle. Cell suspensions were then centrifuged at 110 x 'g' for 10 minutes, the supernatant removed and replaced by a small volume of fresh fixative. The cell pellets were again resuspended by repeated aspiration through a Pasteur pipette.
The final cell suspension dropped (3 to 4 drops per slide) onto the slides. Two slides were prepared from each culture and left to air-dry at room temperature.
- Number of cells spread and analysed per concentration: Approximately 100 metaphase figures from each culture (where possible) with normally a maximum of 25 metaphase figures/slide.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Mitotic index (MI)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Chromosome aberration

Evaluation criteria:

Comparison with solvent control. No further evaluation criteria reported.

Statistics:

- Fisher's test: To compare numbers of aberrant metaphases in each group with the negative control value.
- Yate's test: For any apparent dose-related trend analysis.

Key result

Species / strain:

lymphocytes: obtained from human blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: Based on the range-finding study a top dosage of 4.0 µg/mL without metabolic activation and 8.0 µg/mL with metabolic activation was used for the metaphase analysis as these concentrations caused approximately a 50% reduction in mitotic index.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : Please refer to Table 1.

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements: Please refer to Table 1.
- Genotoxicity results: Please refer to Table 1 .

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 August 2019 to 30 September 2019

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 using the Hprt and xprt genes)

Version / remarks:

29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

August 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines: “Kanpoan No. 287 -- Environment Protection Agency“ “Eisei No. 127 -- Ministry of Health & Welfare“ “Heisei 09/10/31 Kikyoku No. 2 -- Ministry of International Trade & Industry“.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

- Batch no.: 190016P040
- Expiry Date: 06 February 2021
- Storage Conditions: At room temperature, light protected

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: V79 cell line, Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany
- Suitability of cells: High proliferation rate and a good cloning efficiency of untreated cells (as a rule more than 50%)

For cell lines:
- Absence of Mycoplasma contamination: screened
- Methods for maintenance in cell culture: stock cultures were propagated at 37 °C in 75 cm2 plastic flasks. About 2-3×106 cells were seeded into each flask with 15 mL of MEM. The cells were sub-cultured once or twice weekly.
- Doubling time: 12 - 16 h in stock cultures
- Modal number of chromosomes: 22
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature:
MEM: (minimal essential medium) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1%).
All incubations were done at 37°C with 1.5% carbon dioxide (CO2) in humidified air.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Phenobarbital/β-naphthoflavone induced rat liver
- method of preparation of S9 mix: An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4).
- concentration or volume of S9 mix and S9 in the final culture medium: 50 µL/mL
- quality controls of S9: metabolic capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.

Test concentrations with justification for top dose:

0.4; 0.8; 1.6; 3.1; 6.3; 12.5; 25.0; 50.0; 100.0 µg/mL without S9 mix
3.9, 7.8; 15.6; 31.3; 62.5; 125.0; 250.0; 500.0; 750.0 µg/mL with S9 mix

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.
- Justification for percentage of solvent in the final culture medium: 0.5 % (v/v)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 1.5 million cells were seeded in 25 cm² flasks
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 hours
- Harvest time: immediately after the end of treatment

FOR GENE MUTATION:
- Expression time: 7 days
- Selection time: 8 to 11 days
- If a selective agent is used: 6-thioguanine (11 μg/mL) for 8 to 11 days.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: Colonies were stained with 10% methylene blue in 0.01% KOH solution. Colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Cloning efficiency

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The mutant frequency is determined by seeding known numbers of cells in medium containing the selective agent to detect mutant cells, and in medium without selective agent to determine the surviving cells. After a suitable period the colonies are counted. Mutant frequencies are calculated from the number of mutant colonies corrected for cell survival.

Rationale for test conditions:

The dose range of the main experiment was set according to data generated in the pre-experiment. The individual concentrations were spaced by a factor of 2.0 for the experimental part without metabolic activation, closer concentrations were chosen at the highest concentrations for the experimental part with metabolic activation.
To overcome problems with possible deviations in toxicity the main experiment was started with more than four concentrations.
The cultures treated with 25 μg/mL and above (without S9 mix) and at 500 μg/mL and above (with S9 mix) were not continued due to strong toxic effects. The cultures at the lowest concentration of 3.9 μg/mL without S9 mix were not continued as a minimum of only four analysable concentrations is required by the guidelines.

Evaluation criteria:

Acceptability of the Assay
The gene mutation assay is considered acceptable if it meets the following criteria:
a) The mean values of the numbers of mutant colonies per 106 cells found in the solvent controls of both parallel cultures remain within the 95% confidence interval of the laboratory historical control data range.
b) Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistical significant increase compared with the concurrent solvent control.
c) Two experimental conditions (i.e. with and without metabolic activation) were tested unless one resulted in positive results.
d) An adequate number of cells and concentrations (at least four test item concentrations) are analysable even for the cultures treated at concentrations that cause 90% cytotoxicity during treatment.
e) The criteria for the selection of the top concentration are fulfilled.

Interpretation of Results
A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).

A test item is classified as clearly non-mutagenic if, in all experimental conditions examined, all of the following criteria are met:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).

Statistics:

A linear regression (least squares, calculated using a validated excel spreadsheet) will be performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies (mean values) obtained for the groups treated with the test item will be compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: no effect
- Data on osmolality: no effect
- Possibility of evaporation from medium: no
- Precipitation and time of the determination: The test medium was checked for precipitation or phase separation at the beginning and at the end of treatment (4 hours) prior to removal to the test item. Phase separation occurred at 125.0 μg/mL and above after 4 hours treatment without metabolic activation, and at 500.0 μg/mL and above with metabolic activation.

RANGE-FINDING STUDY
In the pre-test approximately 1.5 million cells were seeded in 25 cm² flasks 24 hours prior to treatment. After approximately 24 hours the test item was added and the treatment proceeded for 4 hours (duplicate cultures per concentration level). Immediately after treatment the test item was removed by rinsing with PBS. Subsequently, the cells were trypsinized and suspended in complete culture medium. After an appropriate dilution the cell density was determined with a cell counter. Toxicity of the test item is evident as a reduction of the cell density compared to a corresponding solvent control. A cell density of approximately 1.5 million cells in 25 cm² flasks is about the same as approximately 10 million cells seeded in 175 cm² bottles 24 hours prior to treatment with the main experiment.
The pre-experiment was performed in the presence and absence (4 h treatment) of metabolic activation. Test item concentrations between 15.6 μg/mL and 2000 μg/mL were tested.
In the pre-experiment a relevant cytotoxic effect, indicated by a relative cloning efficiency of 50% or below was observed at 125.0 μg/mL and above without metabolic activation and at 500.0 μg/mL and above with metabolic activation.

STUDY RESULTS
For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible : The linear regression analysis showed no significant dose dependent trend of the mutation frequency at any of the experimental groups.
- Statistical analysis: mean p-value withput S9 mix: 0.884; mean p-value with S9 mix: 0.598

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative survival (RS) and cloning efficiency

- Concurrent vehicle negative and positive control data: Please refer to attached background material
- Genotoxicity results: Please refer to attached background material

HISTORICAL CONTROL DATA
- Positive and negative (solvent) historical control data: Please refer to attached background material

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Results of studies with reliabilities 1 and 2:

In vivo cytogenicity

- Micronucleus in vivo, CAS 42978-66-5 mice, ip, OECD 474: negative (BASF, 2004)

- Micronucleus in vivo, CAS 42978-66-5 mice, oral, OECD 474: negative (Cytec, 2007)

- Micronucleus in vivo, CAS 28961-43-5 mice, oral, OECD 474: negative (BASF, 2002)

- Micronucleus in vivo, CAS 28961-43-5 mice, ip, OECD 474: negative (Cray Valley, 2001)

- Micronucleus in vivo, CAS 28961-43-5 mice, ip, OECD 474: negative (Cytec, 2006)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Specific details on test material used for the study:

- Lot/batch No.: B 02011
- Stability under test conditions: the stability of the test substance under storage conditions is guaranteed as indicated by the sponsor.
- Storage condition of test material: room temperature, protected from light and moisture

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland GmbH
- Age at study initiation: 5-8 weeks
- Weight at study initiation: about 29 g
- Assigned to test groups randomly: yes, under following basis: randomized plan prepared with an appropriate computer program
- Housing: Makrolon cages, type MI, housed individually from start of the treatment until the end of the test
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: Due to the hydrolytical sensitivity of the test substance in water, DMSO was selected as the vehicle, which
had been demonstrated to be suitable in the in vivo micronucleus test and for which historical data are available.
- Concentration of test material in vehicle: 12.5 g/100 ml, 25.0 g/100 ml and 50.0 g/100 ml.
- Amount of vehicle: 4 ml/kg bw.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
All test substance formulations were prepared immediately before administration.
The amount of substance or volume to be administered was related to the specific weight of the individual animals on the day of the experiment.

Duration of treatment / exposure:

single dose

Frequency of treatment:

once, single dose

Post exposure period:

24 or 48 hours

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 animals per dose for the 24 hour sacrifices and 5 animals per dose for the 48 hour sacrifices

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide (CPP) and vincristine sulphate (VCR)
- Justification for choice of positive controls: The stability of CPP and VCR is well-defined under the selected conditions, since both positive control
articles are well-defined clastogens and aneugens respectively.
- Route of administration: The positive controls, both, dissolved in purified water were administered to male animals once orally or intraperitoneally
each in a volume of 10 ml/kg body weight.
- Doses / concentrations: 20 mg CPP//kg body weight for clastogenic effects and 0.15 mg VCR/kg body weight for aneugenic effects.

Tissues and cell types examined:

In general, 2000 polychromatic erythrocytes (PCEs) from each of the animals of every test group are evaluated and investigated for micronuclei (MN).
The normochromatic erythrocytes (NCEs) which occur are also scored .

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In a pretest for the determination of the acute oral toxicity, 2000 mg/kg body weight recommended as the highest dose according to the OECD
Guideline were survived by all animals (male and female) without any clinical signs. Thus, only male animals were used for the cytogenetic
investigations. Therefore, a dose of 2000 mg/kg body weight was selected as the highest dose in the present cytogenetic study. 1000 mg/kg and 500 mg/kg body weight were administered as further doses.

TREATMENT AND SAMPLING TIMES:
The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the híghest dose group of
2000 mg/kg body weight and in the vehicle controls. In the test groups of 1000 mg/kg and 500 mg/kg body weight and in the positive control groups,the 24-hour sacrifice interval was investigated only.

DETAILS OF SLIDE PREPARATION:
The two femora were prepared by dissection and removing all soft tissues. After cutting off the epiphyses, the bone marrow was flushed out of the
diaphysis into a centrifuge tube using a cannula filled with fetal calf serum which was at 37°C (about 2 ml/femur). The suspension was mixed
thoroughly with a pipette, centrifuged at 300 x g for 5 minutes, the supernatant was removed and the precipitate was resuspended in about 50 µl fresh FCS. One drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained.
The slides were stained in eosin and methylene blue solution for 5 minutes (May Grünwald solution modified = Wrights solution), rinsed in purified
water and then placed in fresh purified water for 2 or 3 minutes. They were finally stained in 7.5% Giemsa solution for 15 minutes.
After being rinsed twice in purified water and clarified in xylene, the preparations were mounted using Corbit-Balsam.

METHOD OF ANALYSIS:
In general, 2,000 polychromatic erythrocytes (PCEs) from each of the animals of every test group are evaluated and investigated for micronuclei (MN).
The normochromatic erythrocytes (NCEs) which occur are also scored. The following parameters are recorded:
- Number of polychromatic erythrocytes
- Number of polychromatic erythrocytes containing micronuclei
The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the solvent control group provides an
index of a chromosome-breaking (clastogenic) effect or of a spindle activity of the substance tested.
- Number of normochromatic erythrocytes
- Number of normochromatic erythrocytes containing micronuclei
The number of micronuclei in normochromatic erythrocytes at the early sacrifice intervals shows the situation before test substance administration andmay serve as a control value. A substance-induced increase in the number of micronuclei in normocytes may be found with an increase in the duration of the sacrifice intervals.
- Ratio of polychromatic to normochromatic erythrocytes
An alteration of this ratio indicates that the test substance actually reached the target. Individual animals with pathological bone marrow depression
may be identified and excluded from the evaluation.
- Number of small micronuclei (dD/4) (d = diameter of micronucleus, D= cell diameter)
The size of micronuclei may indicate the possible mode of action of the test substance, i .e . a clastogenic or a spindle poison effect.
Slides were coded before microscopic analysis.
Since the absolute values shown have been rounded off but the calculations were made using the unedited values, deviations in the given relative
values can occur.

Evaluation criteria:

The mouse micronucleus test is considered valid if the following criteria are met:
- The quality of the slides allowed the identification and evaluation of a sufficient number of analyzable cells, i .e. >=2000 polychromatic erythrocytes.
- The proportion of cells with micronuclei in negative control animals was within the normal range of the historical control data.
- The two positive control chemicals induced a significant increase in the number of cells containing small and large micronuclei within the range of
the historical control data or above.

The test chemical is considered positive in this assay if the following criteria are met:
- A dose-related and significant increase in the number of micronucleated polychromatic erythrocytes at any of the intervals.
- The proportion of cells containing micronuclei exceeded both the values of the concurrent negative control range and the negative historical control range.
A test substance is generally considered negative in this test system if:
- There was no significant increase in the number of micronucleated polychromatic erythrocytes at any dose above concurrent control frequencies and at any time.
- The frequencies of cells containing micronuclei were within the historical control range.

Statistics:

The statistical evaluation of the data was carried out using the program system MUKERN.
The number of micronuclei in polychromatic erythrocytes was analyzed.
A comparison of the dose group with the vehicle control was carried out using the Wilcoxon test for the hypothesis of equal medians . Here, the relative frequencies of cells with micronuclei of each animal were used. If the results of this test were significant, labels (* for p <=0.05, ** for p<=0.01) were printed with the group means in the tables. This test was performed one-sided.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Single oral administration of the test substance did not result in any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always close to the range as that of the concurrent negative control and within the range of historical control data. No inhibition of erythropoesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected. According to the authors, the test substance did not have any chromosome-damaging (clastogenic) effect, and there was no indication of any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Specific details on test material used for the study:

- Batch no.: CLX 598/1
- Expiry date: 12 May 1999

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 5-7 weeks old
- Weight at study initiation: 27.2-34.8
- Housing: housed in labelled polycarbonate cages containing purified sawdust
- Diet (e.g. ad libitum): standard pelleted laboratory animal diet; ad libitum
- Water (e.g. ad libitum):tap-water; ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3°C
- Humidity (%): 30-70%.
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours dark per day

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Concentration of test material in vehicle: 250mg/10ml; 125mg/10ml and 62.5mg/10ml

Details on exposure:

The mice received a single intraperitoneal injection of a maximum tolerated (high), an intermediate and a low dose of PURIFIED TMPEOTA. The route of administration was chosen to maximize the chance of the test article reaching the target tissue. The dosing volume was 10 ml/kg body weight.

Duration of treatment / exposure:

single dose

Frequency of treatment:

once, single dose

Post exposure period:

toxicity signs once a day,
24h, 48h treatment groups
48h positiv conrol

Dose / conc.:

62.5 mg/kg bw/day (nominal)

Dose / conc.:

125 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5
two groups per dosage

Control animals:

yes, concurrent vehicle

Positive control(s):

- positiv control: cyclophosphamide
- Route of administration: intraperitoneal
- Doses / concentrations: 50mg/kg bw

Tissues and cell types examined:

The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes.

Details of tissue and slide preparation:

The slides were automatically stained using the "Wright-stain-procedure" in an "Ames" HEMA-tek slide stainer (Miles, Bayer Nederland B.V.). The dry slides were dipped in xylene before they were embedded in MicroMount and mounted with a coverslip.

Evaluation criteria:

The test substance is considered positive in the micronucleus test if:
It Induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test; two-sided test at P < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) in the combined data for both sexes or in the data for male or female groups separately.
A test substance is considered negative in the micronucleus test if:
None of the tested concentrations or sampling times showed a statistically significant (P < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes neither in the combined data for both sexes nor in the data for male or female groups separately.

Statistics:

yes, e.G, Wilcoxon Rank Sum Test; two-sided test at P < 0.05

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

lethargic and rough coat; no abnormalities at 62.5 mg/kg bw

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Specific details on test material used for the study:

- Lot/batch No.: 5010121200
- Storage condition of test material: Stored at room temperature in the dark

Species:

mouse

Strain:

other: Crl:CD-l(ICR)BR

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Age at study initiation: 5-8 wks
- Weight at study initiation: 23-30 g
- Housing: Groups of up to seven in solid-floor polypropylene cages with wood-flake bedding
- Diet: Certified Rat and Mouse Diet Code 5LF2, BCM, IPS Ltd., London, UK, ad libitum
- Water: Ad libitum
- Acclimation period: 7 d


ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 25 °C
- Humidity (%): 30-70 %
- Air changes: 15 per h
- Photoperiod: 12 h dark / h light

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: Arachis oil
- Lot/batch no.: SN365

Details on exposure:

Not applicable

Duration of treatment / exposure:

Single treatment

Frequency of treatment:

Single treatment

Post exposure period:

24 h (for 100, 50 and 25 mg/kg bw dose groups); 48 h (for 100 mg/kg bw dose group)

Dose / conc.:

25 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Seven

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Route of administration: Oral
- Doses / concentrations: 50 mg/kg bw

Tissues and cell types examined:

Tissue: Femoral bone marrow
Cell types: Erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on the results of a range-finding toxicity test, the maximum tolerated dose of the test material, 100 mg/kg bw, with the clinical signs of hunched posture and ptosis was selected for use in the main test, with 50 and 25 mg/kg as the lower dose levels.


TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): 24 or 48 h following dosing


DETAILS OF SLIDE PREPARATION: Both femurs were dissected from each animal, aspirated with foetal calf serum and bone marrow smears prepared
following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, stained in May-Grunwald/Giemsa, allowed to air-dry and cover-slipped using mounting medium.


METHOD OF ANALYSIS: Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification. The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCEblue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted; these cells were also scored for incidence of micronuclei. The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.

Evaluation criteria:

A positive mutagenic response was demonstrated when a statistically significant, dose-responsive, toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48 h kill times when compared to their corresponding control group. A positive response for bone marrow toxicity was demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the concurrent vehicle control group.

Statistics:

The data was analysed following a √(x +1) transformation using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

No significant effects except hunched posture and ptosis

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: Maximum tolerated dose: 100 mg/kg bw i.p. No toxicity was observed by oral route, therefore, systemic absorption could not be confirmed using this dose route.
- Clinical signs of toxicity in test animals: In animals dosed with the test material via the intraperitoneal route premature deaths (animals killed in extremis) occurred at and above 200 mg/kg bw, and clinical signs were observed at and above 100 mg/kg bw as follows: Hunched posture, ptosis, lethargy, ataxia, increased salivation, piloerection, decreased respiratory rate, laboured respiration, hypothermia, splayed gait, prostration and elevated tail.
- Other: The test material showed no marked difference in its toxicity to male or female mice; it was therefore considered to be acceptable to use males only for the main test.

RESULTS OF DEFINITIVE STUDY
- Ratio of PCE/NCE (for Micronucleus assay): There were no statistically significant decreases in the PCE/NCE ratio in the 24 or 48 h test material groups when compared to their concurrent vehicle control groups. However, the observation of clinical signs was taken to indicate that systemic absorption had occurred. There were no statistically significant increases in the frequency of micronucleated PCEs in any of the dose groups when compared to their concurrent vehicle control groups.
- Other: Mortality data and clinical observations: There were no premature deaths seen in any of the dose groups. Following clinical signs were observed in animals dosed with the test material at 100 mg/kg bw in both the 24 and 48 h groups: Hunched posture and ptosis.

Table 1: Micronucleus Test - Summary of Group Mean Data

Treatment Group	Number of PCE with Micronuclei per 2000 PCE		PCE/NCE Ratio
	Group Mean	SD	Group Mean	SD
Vehicle Control (Arachis oil) 10 mL/kg bw 48 h Sampling Time	1.1	1.2	0.83	0.38
Vehicle Control (Arachis oil) 10 mL/kg bw 24 h Sampling Time	1.1	1.2	0.76	0.23
Positive Control 50 mg/kg bw 24 h Sampling Time	86.4***	27.8	1.35	0.27
Test substance 100 mg/kg bw 48 h Sampling Time	1.3	0.8	0.9	0.33
Test substance 100 mg/kg bw 24 h Sampling Time	1.4	1.9	0.78	0.34
Test substance 50 mg/kg bw 24 h Sampling Time	0.1	0.4	0.89	0.26
Test substance 25 mg/kg bw 24 h Sampling Time	1.9	1.6	0.71	0.51

where,

PCE = Polychromatic erythrocytes

NCE = Normochromatic erythrocytes

SD = Standard deviation

*** = P < 0.001

Reference 4

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2003-11-07 to 2004-03-08

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:

July 21, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

May 19, 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Specific details on test material used for the study:

- Batch number of test material: 030061P040
- Expiration date of the batch: not available, but stability of the test substance throughout the study period was guaranteed until April 2004

Species:

mouse

Strain:

NMRI

Remarks:

CrI:NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Germany
- Age at study initiation: 5-8 weeks
- Weight at study initiation: 31 g (mean)
- Assigned to test groups randomly: yes, under following basis: randomization plan prepared with an appropriate computer program
- Housing: Makrolon cages, type MI, housed individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

intraperitoneal

Vehicle:

- Vehicle used: olive oil
- Justification for choice of vehicle: Due to the limited solubility of the test substance in water, olive oil was selected as the vehicle, which had been demonstrated to be suitable in the in vivo micronucleus test and for which historical data are available.
- Concentration of test material in vehicle: 0.875 g/100 mL, 1.75 g/100 mL and 3.5 g/100 mL
- Amount of vehicle: 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The substance to be administered per kg body weight was dissolved in olive oil immediately before administration:
- The low dose group was given 87.5 mg test substance/kg body weight or 10 mL/kg body weight of a solution with a concentration of
0.875 g/100 mL.
- The intermediate dose group was given 175 mg test substance/kg body weight or 10 mL/kg body weight of a solution with a concentration of
1.75 g/100 mL.
- The top dose groups were given 350 mg test substance/kg body weight or 10 mL/kg body weight of a solution with a concentration of 3.5 g/100 mL

Duration of treatment / exposure:

one single administration

Frequency of treatment:

one single administration

Post exposure period:

24-48 hours

Dose / conc.:

87.5 mg/kg bw/day (nominal)

Dose / conc.:

175 mg/kg bw/day (nominal)

Dose / conc.:

350 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (CPP) and vincristine (VCR) both dissolved in purified water were administered to male animals once intraperitoneally each in a volume of 10 mL/kg body weight.
- Justification for choice of positive controls: The stability of CPP and VCR is well-defined under the selected conditions, since both positive control articles are well-established reference clastogens and aneugens respectively.
- Route of administration: intraperitoneal
- Doses / concentrations: CPP: 20 mg/kg bw for clastogenic effects; VCR: 0.15 mg/kg bw for aneugenic effects

Tissues and cell types examined:

In general, 2000 polychromatic erythrocytes (PCEs) from each of the animals of every test group are evaluated and investigated for micronuclei (MN).
The normochromatic erythrocytes (NCEs) which occur are also scored.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
To determine the doses a pretest was conducted. In the pretest for the determination of the acute intraperitoneal toxicity, deaths were observed down to a dose of 500 mg/kg body weight. 250 mg/kg body weight were survived by all animals (male and female), but led to clinical signs such as piloerection and squatting posture. However, there were no distinct differences in the symptoms between males and females. Thus, only male animals were used for the cytogenetic investigations.
Therefore, a dose of 350 mg/kg body weight was selected as the highest dose in the present cytogenetic study. 175 mg/kg and 87.5 mg/kg body weight were administered as further doses.

TREATMENT AND SAMPLING TIMES:
The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of 350 mg/kg body weight and in the vehicle controls. In the test groups of 175 mg/kg and 87.5 mg/kg body weight and in the positive control groups the 24-hour sacrifice interval was investigated only.

DETAILS OF SLIDE PREPARATION:
The two femora were prepared by dissection and removing all soft tissues. After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum which was at 37 °C (about 2 mL/femur). The suspension was mixed thoroughly with a pipette, centrifuged at 300 x g for 5 minutes, the supernatant was removed and the precipitate was resuspended in about 50 µL fresh FCS. One drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained.
The slides were stained in eosin and methylene blue solution for 5 minutes (May Grünwald solution modified = Wrights solution), rinsed in purified water and then placed in fresh purified water for 2 or 3 minutes. They were finally stained in 7.5 % Giemsa solution for 15 minutes. After being rinsed twice in purified water and clarified in xylene, the preparations were mounted using Corbit-Balsam.

METHOD OF ANALYSIS:
In general, 2000 polychromatic erythrocytes (PCEs) from each of the animals of every test group were evaluated and investigated for micronuclei (MN).
The normochromatic erythrocytes (NCEs) which occur were also scored. The following parameters were recorded:
- Number of polychromatic erythrocytes
- Number of polychromatic erythrocytes containing micronuclei
The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the solvent control group provides an index of a chromosome-breaking (clastogenic) effect or damage of the mitotic apparatus (aneugenic activity) of the substance tested.
- Number of normochromatic erythrocytes
- Number of normochromatic erythrocytes containing micronuclei
The number of micronuclei in normochromatic erythrocytes at the early sacrifice intervals shows the situation before test substance administration and may serve as a control value. A substance-induced increase in the number of micronuclei in normocytes may be found with an increase in the duration of the sacrifice intervals.
- Ratio of polychromatic to normochromatic erythrocytes
An alteration of this ratio indicates that the test substance actually reached the target. Individual animals with pathological bone marrow depression may be identified and excluded from the evaluation.
- Number of small micronuclei (d=D/4) (d = diameter of micronucleus, D= cell diameter)
The size of micronuclei may indicate the possible mode of action of the test substance, i .e . a clastogenic or a spindle poison effect.
Slides were coded before microscopic analysis.
Since the absolute values shown have been rounded off but the calculations were made using the unedited values, deviations in the given relative values can occur.

Evaluation criteria:

The mouse micronucleus test was considered valid if the following criteria were met:
- The quality of the slides allowed the identification and evaluation of a sufficient number of analyzable cells, i .e. >=2000 polychromatic erythrocytes and a clear differentiation between polychromatic erythrocytes (PECs) and normochromatic erythrocytes (NECs).
-The ratio of PECs/NECs in the untreated animals (negative control) has to be within the normal range of the animal strain.
- The number of cells containing micronuclei in negative control animals has to be within the range of the historical control data both for PECs and NECs.
- The two positive control substances have to induce a significant increase in the number of PECs containing small and large micronuclei within the range of the historical control data or above.

A finding was considered positive if the following criteria were met:
- Significant and dose-related increase in the number of PCEs containing micronuclei.
- The number of PCEs containing micronuclei has to exceed both the concurrent negative control and the highest value of the historical control range.

A test substance was considered negative if the following criteria were met:
- The number of cells containing micronuclei in the dose groups was not significantly above the negative control and was within the historical control data.

Statistics:

The statistical evaluation of the data was carried out using the program system MUKERN.
The asymptotic U test according to Mann-Whitney (modified rank test according to Wilcocon) was carried out to clarify the question whether there were significant differerences between the control group and dose groups with regard to the micronucleus rate in polychromatic erythrocytes. The relative frequencies of cells containing micronuclei of each animal was used as a criterion for the rank determination for the U test.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

The test substance led to clinical signs in the animals and dose-dependent inhibition of erythropoiesis induced by the treatment was detected from about of 87.5 mg/kg body weight onward.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: Based on the pretest 350 mg/kg body weight was selected as the highest dose for the main study.
- Solubility: soluble in the vehicle selected
- Clinical signs of toxicity in test animals: Deaths were observed down to 500 mg/kg bw. At 250 mg/kg bw piloerection and squatting posture were observed.
- Other: The pretest examined the acute intraperitoneal toxicity to animals and did not determine any further parameters.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei : The test substance did not lead to any increase in the rate of micronuclei (small and large). For numbers please refer to the attached result tables.
- Ratio of PCE/NCE: A dose-dependent inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected from about of 87.5 mg/kg body weight onward.

Reference 5

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2007-04-03 to 2007-08-14

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:

21 July 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Specific details on test material used for the study:

- Lot number of test material: P8960464SAP
- Expiration date of the lot: 2008-03-19

Species:

mouse

Strain:

CD-1

Remarks:

CD-1(ICR) BR

Details on species / strain selection:

The strain is an outbred strain that maximizes genetic heterogeneity and therefore tends to eliminate strain-specific response to test articles.
The mouse has been routinely utilized as an animal model of choice for the mammalian bone marrow erythrocyte micronucleus assay.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan, Frederick, Maryland, USA
- Age at study initiation: Young adults, approx. 8 weeks old
- Weight at study initiation: males: 33.7 to 38.2 g; females: 23.1 to 28.1 g
- Assigned to test groups randomly: yes, by using a computer program
- Housing: separated by gender, up to 5 animals per cage
- Diet: PMI Certified Rodent Diet (R) #5002, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 17.7 - 26.1 °C
- Humidity: 30-70 %
- Air changes per hr: >/= 10
- Photoperiod: 12 hrs dark / 12 hrs light

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: Corn oil (CAS No. 8001-30-7)
- Justification for choice of vehicle: In water application, the test article was observed to form a white, opaque, homogeneous, liquid emulsion. In corn oil, the test article was observed to form a slightly viscous, transparent, light-yellow solution. As the test material was successfully suspended in corn oil, it was selected as the vehicle.
- Concentration of test material in vehicle: 50, 100 and 200 mg/mL
- Amount of vehicle: 10 mL/kg bw
- Lot No.: 12-455, by Supplier Welch, Holme & Clarke

Details on exposure:

Dose Preparation
Prior to dosing, the top stock of the test article was prepared by adding the appropriate volume of the vehicle, corn oil, to a pre-weighed quantity of the test article and mixed, forming a solution. Lower concentrations were obtained by dilution with the vehicle. The formulations were held at room temperature prior to dosing.

Duration of treatment / exposure:

one single administration

Frequency of treatment:

one single administration

Post exposure period:

24 h for all dose groups; additionally 48 h for an additional vehicle control and an additional 2000 mg/kg bw group

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Range-finding study: 3 males and 3 females per dose
Main study: 5 males per dose and harvest time point

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (CAS No. 6055-19-2); Supplier: Sigma Aldrich; Lot No. 076K1050; Storage in a refrigerator set to maintain >0 to 10 °C;
- Route of administration: oral gavage
- Doses / concentrations: 8 mg/mL in water, 10 mL/kg bw were administered

Tissues and cell types examined:

Bone marrow
polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Since no appropriate toxicity data were available (e.g., the same species, strain, same route, etc.), a dose range-finding study was performed using both male and female animals (to determine if there were any sex-related differences in toxicity). The daily observations of toxic signs and/or mortality data were used to estimate the highest appropriate dose level (maximum tolerated dose) for the micronucleus assay. The test article was formulated in corn oil and administered once by oral gavage to 3 males and 3 females per dose level for dose levels of 500, 1000, and 2000 mg/kg bw. The animals were observed for up to 2 days after the last dose administration for toxic signs and/or mortality. Based on the results of the dose range-finding study, the high dose chosen was 2000 mg/kg bw, the limit dose based on regulatory guidelines.

DETAILS OF SLIDE PREPARATION:
Extraction of Bone Marrow
The hind limb bones (tibias) were removed for marrow extraction from five surviving animals in each treatment and control group. For each animal, the marrow flushed from the bones was combined in an individual centrifuge tube containing 3 to 5 mL fetal bovine serum (one tube per animal).
Preparation of Slides
Following centrifugation to pellet the marrow, the supernatant was removed by aspiration and portions of the pellet were spread on slides and air-dried. The slides were fixed in methanol, stained in May-Grünwald solution and Giemsa, and protected by mounting with coverslips. For control of bias, all slides were coded prior to analysis.

METHOD OF ANALYSIS:
Slides prepared from the bone marrow collected from five animals per group at the designated harvest timepoints were scored for micronuclei and the PCE to NCE cell ratio. The micronucleus frequency (expressed as percent micronucleated cells) was determined by analyzing the number of micronucleated PCEs from at least 2000 PCEs per animal. The PCE:NCE ratio was determined by scoring the number of PCEs and NCEs observed while scoring at least 500 erythrocytes per animal.

Evaluation criteria:

Acceptable Controls
The vehicle control group mean must lie within the historical control range and will usually be less than 0.4 % micronucleated PCEs. There must be a statistically significant elevation of the mean of the positive control group relative to the vehicle control group, and the positive control response must be consistent with historical positive control data.
Acceptable High Dose
Generally the high dose should reach the limit dose or produce some indication of toxicity, e.g., toxic signs and/or mortality in the test article dosed animals and/or a reduction in the PCE:NCE ratio. If there are solubility constraints, the highest dose tested will be the solubility limit or higher doses if a well-dispersed suspension is obtained that does not settle out rapidly.
Assay Evaluation Criteria
The criteria for a positive response is the detection of a statistically significant increase in micronucleated PCEs for at least one dose level, and a statistically significant dose related response. A test article that does not induce both of these responses is considered negative. Statistical significance is not the only determinant of a positive response; the Study Director also considers the biological relevance of the results in the final evaluation.

Statistics:

The following statistical methods were used to analyze the micronucleus data.
- Assay data analysis was performed using an analysis of variance (Winer, 1971) on untransformed proportions of cells with micronuclei per animal and on untransformed PCE:NCE ratios when the variances were homogeneous. Ranked proportions were used for heterogeneous variances.
- If the analysis of variance was statistically significant (p ≤ 0.05), Dunnett's t-test (Dunnett, 1955; 1964) was used to determine which dose groups, if any, were statistically significantly different from the vehicle control. Analyses were performed separately for each sampling time.
The 500, 1000, and 2000 mg/kg dose groups, as well as the positive control group, were compared with the vehicle control group at the 5% probability level.

Key result

Sex:

male

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: The high dose of the test substance reached the maximum allowable dose of 2000 mg/kg bw, based on regulatory guidelines.
- Solubility: Solubility testing was performed in two vehicles. The test substance formed emulsion with water but desolved in corn oil. Therefore, corn oil was used as vehicle.
- Clinical signs of toxicity in test animals: All animals appeared normal immediately after dosing and remained healthy until the end of the observation period.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: The test substance did not induce statistically significant increases in micronucleated PCEs at any test article dose examined (500, 1000, or 2000 mg/kg). For numbers please refer to the attached result tables.
- Ratio of PCE/NCE: The test substance was not cytotoxic to the bone marrow (i.e., no statistically significant decreases in the PCE:NCE ratios) at any dose of the test article analyzed. For numbers please refer to the attached result tables.
- Appropriateness of dose levels and route: The maximum allowable dose of 2000 mg/kg bw, based on regulatory guidelines, was used.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro

 

Gene mutation in bacteria

The substance was tested for its ability to induce point mutations in a reverse mutation assay according to OECD guideline 471 (BASF, 2004). The strains TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA were tested with a dose range of 20 µg - 5 000 µg/plate (SPT) and 20 µg - 5 000 µg/plate (PIT). Both test conditions, Standard plate test (SPT) and preincubation test (PIT) were performed with and without metabolic activation (Aroclor-induced rat liver S-9 mix). A weak bacteriotoxicity effect was occasionally observed depending on the strain and test conditions at doses ≥ 2 500 µg/plate. An increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S-9 mix or after the addition of a metabolizing system.

 

In a second study performed similar to OECD guideline 471 (Cytec, 1983), the test substance was added to the cells both in the presence and absence of a liver microsomal fraction. The following strains were tested: S. typhimurium TA 1535 his G46, S. typhimurium TA 1537 his C3076, S. typhimurium TA 1538 his D3052,S. typhimurium TA 98 his D3052, S. typhimurium TA 100 his G46. All five strains are defective in DNA repair capacity (∆uvr B-). The test substance was not toxic towards the tester strains. Therefore 5000 µg/plate was chosen as the top dose level in the mutation tests. No substantial increases in the revertant colony numbers of any of the five strains were observed following treatment with the test substance at any dose level, either in the presence or absence of liver microsomal fraction (S-9 mix).

 

The test substance was tested in the Salmonella/mammalian-microsome mutagenicity assay using five tester strains, TA98, TA100, TA1535, TA1537 and TA1538, both with and without metabolic activation by Aroclor induced rat liver microsomes (Cognis, 1982). The test was performed according to the protocol of Ames et al. The results of the preliminary toxicity determination indicate that no appreciable toxicity was observed up to the 10,000 µg per plate dose level. The results of the Salmonella/mammalian-microsome mutagenicity assay indicate that the test article did not cause a positive response on any of the tester strains with or without metabolic activation by Aroclor induced rat liver microsomes.

 

In another study similar to OECD guideline 471 the test substance was tested directly and in the presence of liver microsomal enzyme preparations from Aroclor-induced rats in several Salmonella strains (Cytec, 1984). Based on results of a preliminary study in S. typhimurium TA-100 cells, 8 doses of 0.1 to 150.00 µL (~mg) per plate were added to the tester strains S. typhimurium TA 1538, TA 1535, TA 1537, TA 98 and TA 100. Toxicity to TA-100 occurred at 75 and 150 µL per plate in the pre-test. The test substance was considered non-mutagenic in this assay.

 

In conclusion, the test substance did not cause gene mutations in bacteria in all four available studies.

 

Gene mutation in mammalian cells

A study was performed to investigate the potential of the test substance to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster (ReachCentrum, 2020). The treatment period was 4 hours with and without metabolic activation. The maximum test item concentration of the pre-experiment (2000 μg/mL) was chosen with respect to the current OECD guideline 476 (2016). The highest concentration in the main experiment was limited by phase separation observed in the pre-experiment.

The main experiment was analysed for gene mutation at the following concentrations:

without S9 mix: 0.4; 0.8; 1.6; 3.1; 6.3; and 12.5* μg/mL

with S9 mix: 7.8; 15.6; 31.3; 62.5; 125.0; and 250.0* μg/mL

*Data at this concentration are not taken into consideration for mutation analysis due to strong cytotoxic effects (rel. adjusted CE I below 10%)

Phase separation was observed at 12.5 μg/mL in the absence of metabolic activation, and at 250.0 μg/mL in the presence of metabolic activation. A relevant cytotoxic effect indicated by the mean relative adjusted cloning efficiency I below 50% was observed at a concentration of 6.3 μg/mL and above in the absence of metabolic activation. At the highest concentration of 12.5 μg/mL the relative adjusted cloning efficiency I was far below 10% (4.8%). Based on the OECD guideline 476, positive mutation findings should be considered with care in case of cytoxicity below 10%. Therefore, the data at 12.5 μg/mL without S9 mix are not taken into consideration for mutation analysis. In the presence of metabolic activation relevant cytotoxicity was observed at 125.0 μg/mL. At the highest concentration of 250.0 μg/mL the relative adjusted cloning efficiency I was below 10% as well (7.1%). Consequently the data at this concentration were not taken into consideration for mutation analysis. No substantial and dose dependent increase of the mutation frequency was observed in the main experiment. EMS (300 μg/mL) and DMBA (2.3 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the substance is considered to be non-mutagenic in this HPRT assay.

The following two studies are of lesser reliability (Val. 4) and are not considered relevant for risk assessment:

 

Another study was conducted to assess the mutagenic potential of the test substance on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line (Cytec, 1994) according to OECD guideline 476, and EEC Directive 87/302/EEC Part B: Mutagenicity testing and screening for carcinogenicity. Two independent tests each in the absence and presence of exogenous metabolic activation (S-9 mix) were carried out. In the absence of S-9 mix, cells were treated with 0.1, 0.25, 0.5, 1 and 2.5 µg/mL in test 1 and 0.5, 1, 2, and 3 µg/mL in test 2. Cultures were cloned in soft agar to permit measurement of the levels of viability and induced mutation. Significant dose-related, increases in mutant frequency were observed in both tests with increases of greater than twice the control level at 2.5 µg/mL in test 1, and 2 and 3 µg/mL in test 2. All positive concentrations were also cytotoxic. Survival was reduced to 3% in test 1 and to 27 -49% in test 2. The positive control induced highly significant increases in mutant frequency in both tests.

In the presence of S-9 mix, cells were treated with 10, 20, 30 and 40 µg/mL in test 1 and 10, 15, 20, 25 and 30 µg/mL in test 2. They were cloned in soft agar to permit measurement of the levels of viability and induced mutation. Dose-related, statistically significant increases in mutant frequency were observed in test 1 with increases of twice the control level at 40 µg/mL. In test 2, although statistically significant increases in mutant frequency were observed, these were not dose-related nor twice the control level. Cytotoxicity was also observed in both tests. Viability was reduced to 11% in the positive concentration of test 1. Methylcholanthrene, the positive control, induced highly significant increases in mutant frequency in both tests.

The colony size distribution was analysed for all cultures assessed for induced mutation, though no data were provided. In the presence of S-9 mix, no decreases in colony size compared to the controls were observed in either test. However, in the absence of S-9 mix a decrease in colony size was observed in both tests which hints towards a clastogenic effects. In conclusion, the test item was considered to be mutagenic to L5178Y cells with and without metabolic activation.

 

A second mouse lymphoma assay also performed in L5178Y cells provides a comparable result (Litton Bionetics, 1983). Cells were either treated with 0.5, 0.75, 1, 1.5, 2, and 3 µg/mL in the absence of S9 or with 2.5, 15, 20, 30, 50, 60 µg/mL in the presence of S9. Concentrations were selected on a preliminary cytotoxicity assay to yield at least 70 -90% reduction in relative growth in the top dose. A relevant and dose dependent increase occurred at and above 1.5 µg/mL without S9 and at and above 15 µg/mL with S9. All positive concentration also resulted in severe cytotoxicity (relative growth between 17.5% and 4.9% without S9 and between 43.9% and 3.4% with S9) with a steep dose-response, e.g., an increase from 20 µg/mL to 30 µg/mL reduced the relative growth by about a factor of 2. A further increase to 50 µg/ml was almost completely lethal. Positive and negative controls were valid, though cloning efficiencies for the solvent controls were lower than usually acceptable. No information on colony size was provided, so clastogenic activity or point mutations cannot be differentiated based on this report.

 

Chromosome aberration in mammalian cells

Human lymphocytes were exposed to the test item with and without metabolic activation in a study similar to OECD guideline 473 (Cytec, 1983). A preliminary toxicity test was carried out to assess the effect of the test item on the mitotic index of cultured human lymphocytes. The results indicated that a concentration of 4.0 µg/mL without metabolic activation and 8.0 µg/mL with metabolic activation would cause a decrease in mitotic index of approximately 50% (EC50). Human lymphocytes were grown in tissue culture medium at 37°C and mitotic cell division was stimulated by the addition of phytohaemaglutinin. For metaphase analysis without metabolic activation the test item was added to reach final concentrations of 0.4, 2.0 and 4.0 µg/mL after 48 h incubation. The cultures were incubated for a further 24 h. DMSO and sterile distilled water were used as the solvent controls and mitomycin C (0.4 µg/mL) and ethylmethane sulphonate (500 µg/mL) were used as the positive controls. For metaphase analysis with metabolic activation, S-9 mix was added at a final concentration of 250 µg/mL followed by the test item at final concentrations of 0.8, 4.0 and 8.0 µg/mL. The cultures were incubated for 2 h and the cells were then harvested, washed and resuspended in fresh tissue culture medium. The cultures were incubated for a further 22 h. The positive control agent used was cyclophosphamide (20 µg/mL). DMSO and sterile distilled water were used as the solvent controls.

The lymphocytes were arrested in the metaphase stage of cell division, fixed, stained and examined microscopically for evidence of chromosomal damage. Cultures treated with the test item did not cause any increase in the amount of detectable chromosomal damage when compared with the solvent control treated cultures either in the presence or absence of metabolic activation. The results of positive control compounds confirmed the sensitivity of the test system to the effects of a direct acting and indirect acting mutagen. In conclusion, the test item did not cause chromosome aberrations in human lymphocytes with and without metabolic activation.

 

Conclusion

The test substance caused a dose dependent increase in the number of mutant colonies in two mouse lymphoma assays with and without S9 and highly cytotoxic concentrations. Based on the limited data available from the first study, this effect is more likely caused by clastogenicity rather than the ability to cause point mutations. This result does not contradict the negative chromosome aberration assay, since survival in this assay was only reduced by a maximum of 50%, a condition that also did not cause a positive response in the mouse lymphoma assays. In addition, neither the Ames tests nor the HPRT test revealed a positive response indicative of a mutagenic potential of the test item. Furthermore, a publication by Johannsen et al. (Regulatory Toxicology and Pharmacology 50 (2008) 322–335) suggests, that the mouse lymphoma assay is not the ideal test system for testing acrylates for their gene mutation potential in mammalian cells.

 

In vivo

 

No in vivo mammalian somatic cell study is available for the substance to be registered (target substance). Therefore, a read-across approach to three different structural analogues is used to address this endpoint. 

The studies are used in a weight of evidence approach to evaluate the target substance in regard to its potential for clastogenicity and aneugenic activity.

 

Two studies according to OECD 474 were conducted with the read-across substance CAS 42978-66-5.

 

The read-across substance CAS 42978-66-5, dissolved in olive oil, was administered once intraperitoneally to male animals (5 per group) at dose levels of 87.5 mg/kg, 175 mg/kg and 350 mg/kg body weight in a volume of 10 mL/kg body weight in each case (BASF AG, 2004). The administered doses were determined based on a pretest to evaluate the toxicity of the source substance after interperitoneal injection. In the pretest deaths were observed down to a dose of 500 mg/kg body weight. 250 mg/kg body weight were survived by all animals (male and female), but led to clinical signs such as piloerection and squatting posture. However, there were no distinct differences in the symptoms between males and females. Thus, only male animals were used for the cytogenetic investigations. Based on the pretest a dose of 350 mg/kg body weight was selected as the highest dose in the present cytogenetic study and 175 mg/kg and 87.5 mg/kg body weight were administered as further doses. As a negative control, male mice were administered merely the vehicle, olive oil, by the same route, which gave frequencies of micronucleated polychromatic erythrocytes within the historical control range. Both of the positive control chemicals, i .e. cyclophosphamide for clastogenicity and vincristine for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei. Animals which were administered the vehicle or the positive control substances cyclophosphamide or vincristine did not show any clinical signs of toxicity. The administration of the source substance led to clinical signs. The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of 350 mg/kg body weight and in the vehicle controls. In the test groups of 175 mg/kg and 87 .5 mg/kg body weight and in the positive control groups, the 24-hour sacrifice interval was investigated only. After staining of the preparations, 2 000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2 000 polychromatic erythrocytes were also recorded. According to the results of the present study, the single intraperitoneal administration of the source substance did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always close to the range as that of the concurrent negative control in all dose groups and at all sacrifice intervals and within the range of the historical control data. A dose-dependent inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected from about of 87 .5 mg/kg body weight onward. Thus, under the experimental conditions, the read-across substance CAS 42978-66-5 does not have any chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.

 

In another study with application of the read-across substance CAS 42978-66-5 by gavage 5 male animals were treated per dose (Cytec, 2007). In the dose range-finding study, the source substance was formulated in corn oil and administered once by oral gavage to 3 males and 3 females per dose level for dose levels of 500, 1000, and 2000 mg/kg bw. The animals were observed for up to 2 days after the last dose administration for toxic signs and/or mortality. Based on the results of the dose range-finding study, the high dose chosen was 2000 mg/kg bw, the limit dose based on regulatory guidelines. In the micronucleus assay, the test article was formulated in corn oil and administered once orally by gavage at 500, 1000, and 2000 mg/kg bw. Furthermore, a positive control group using cyclophosphamide and vehicle control groups of 5 male animals were treated. Harvest was after 24 h for all doses and in addition after 48 h for vehicle control and the high dose. Bone marrow was extracted and at least 2000 PCEs per animal were analyzed for the frequency of micronuclei. Cytotoxicity was assessed by scoring the number of polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs) in at least the first 500 total erythrocytes for each animal. The source substance did not induce signs of clinical toxicity in the animals treated at dose levels up to 2000 mg/kg bw. The read-across substance CAS 42978-66-5 did not induce statistically significant increases in micronucleated PCEs at any dose examined (500, 1000, and 2000 mg/kg bw). In addition, the source substance was not cytotoxic to the bone marrow (i.e., no statistically significant decreases in the PCE:NCE ratios) at any dose level. Based on these results the source substance was evaluated as negative in the mouse bone marrow micronucleus assay under the conditions of this assay.

 

Three studies according to OECD 474 were conducted with the read-across substance CAS 28961-43-5

 

The read-across substance CAS 28961-43-5 was tested for chromosomal damage (clastogenicíty) and for the ability to induce spindle poison effects (aneugenic activity) in NMRI mice using the micronucleus test method according to OECD guideline 474 (BASF, 2002). For this purpose, the test substance, dissolved in DMSO, was administered once orally to male animals at dose levels of 500 mg/kg, 1,000 mg/kg and 2,000 m g/kg body weight in a volume of 4 ml/kg body weight in each case. As a negative control, male mice were administered merely the vehicle, DMSO, by the same route, which gave frequencies of micronucleated polychromatic erythrocytes within the historical control range. Both of the positive control chemicals, i .e . cyclophosphamide for clastogenicity and vincristine for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei. Animals which were administered the vehicle or the positive control substances cyclophosphamide or vincristine did not show any clinical signs of toxicity. The administration of the test substance was tolerated by all animals without any signs or symptoms. The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of 2,000 mg/kg body weight and in the vehicle controls. In the test groups of 1,000 mg/kg and 500 mg/kg body weight and in the positive control groups, the 24-hour sacrifice interval was investigated only. After staining of the preparations, 2,000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2,000 polychromatic erythrocytes were also recorded. According to the results of the present study, the single oral administration of the test substance did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always close to the range as that of the concurrent negative control in all dose groups and at all sacrifice intervals and within the range of the historical control data. No inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected. Thus, under the experimental conditions chosen, the test substance does not have any chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.

 

In a further micronucleus test in mice according to OECD guideline 474 the read-across substance CAS 28961-43-5 was evaluated for its genotoxic effect on erythrocytes in bone marrow cells (Cray Valley, 2001). Six groups each comprising 5 males and 5 females, received a single intraperitoneal injection. Two groups (A and B) were dosed with 250 mg/kg body weight, two groups (C and D) were dosed with 125 mg/kg body weight and two groups (E and F) were dosed with 62.5 mg/kg body weight. One group (G) treated with a single intraperitoneal injection of cyclophosphamide (CP) at 50 mg/kg body weight served as positive control. No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with the test substance. The animals of the groups treated with 250 mg per kg body weight showed slight decreases in the ratio of polychromatic to normochromatic erythrocytes at the 48 hours sampling time compared to the ratio of polychromatic to normochromatic erythrocytes at the 48 hours sampling time of the animals treated with the lowest dose group of 62.5 mg/kg body weight. This phenomenon reflects a toxic effect of the test substance on the erythropoiesis, implying that the substance reached the bone marrow cells of the mouse. It is concluded that the test item is not mutagenic in the micronucleus test under the experimental conditions described in the report.

 

Another study was performed to investigate the potential of the read-across substance CAS 28961-43-5 to induce micronuclei in the bone marrow of the Crl:CD-l(ICR)BR mouse according to OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test) and EU Method B.12 (Mutagenicity - In vivo Mammalian Erythrocyte Micronucleus Test) (Cytec, 2006). A range-finding test was performed to find suitable dose levels of the test substance, route of administration and to investigate if there was a marked difference in toxic response between the sexes. There was no marked difference in toxicity between the sexes; therefore the main test was performed using only male mice. The micronucleus test was conducted using the intraperitoneal route in groups of seven mice at the maximum tolerated dose of 100 mg/kg bw and with 50 and 25 mg/kg bw as the two lower dose levels. Animals were killed 24 or 48 h later, the bone marrow extracted, and smear preparations made and stained. Polychromatic and normochromatic erythrocytes were scored for the presence of micronuclei. Further groups of mice (each of 7 mice) were given a single intraperitoneal dose of arachis oil or dosed orally with cyclophosphamide (5 mice), to serve as vehicle and positive controls respectively. Vehicle control animals were killed 24 or 48 h later, and positive control animals were killed after 24 h.  

There were no premature deaths seen in any of the dose groups. Hunched posture and ptosis were observed in animals dosed with test substance at 100 mg/kg bw. No statistically significant decreases in the PCE/NCE ratio were observed in the test groups when compared to concurrent control groups. However, the observation of clinical signs was taken to indicate that systemic absorption had occurred. There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in test groups when compared to the concurrent vehicle control groups. The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test. In conclusion, the test substance was considered to be non-genotoxic in the micronucleus test in the mouse.

 

Conclusion

Since all available in vivo cytogenetic studies with the read-across substances revealed a negative result it is concluded that the target substance is neither expected to have any chromosome-damaging (clastogenic) effect, nor to induce an impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available data for genetic toxicity are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on this data, the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) 2019/521.