Oxazolidine, diheptyl~

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

REACH_negative | TA 98, TA 100, TA 1535, TA 1537 and Ecoli WP2 uvr A | OECD 471 | with and without | #key study#

REACH_positive │Human lymphocytes│OECD 473 | with and without | #key study#

REACH_negative | V79 cells | OECD 476 | with and without | #key study#

Sa 34 did not induce gene mutations in Salmonella typhimurium and Escherichia coli strains up to a concentration of 1000 µg/plate in a GLP compliant study according to OECD Technical Guidelines 471 and is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster according GLP compliant study following the OECD Technical Guidelines 476.

 

But Sa 34 induced structural chromosomal aberrations in human lymphocytes without metabolic activation, when performing a GLP compliant study according to OECD Technical Guidelines 473. The aberration rates found at the concentrations of 0.01 µL/mL (4.3 %), 0.02 µL/mL (4.7 %) and 0.05 µL/mL (5.3%) were increased compared to the corresponding negative control (0.3 %) and the historic control range of the testing facility (-0.21 to 3.94%). This increase was not statistically significant. The concentration-related increase was not statistically significant, as well. Thus, only one of the three experimental conditions showing clearly positive substances, is fulfilled (results were outside the distribution of the historical negative control data). Therefore, more tests need to be performed to clarify the result. In order to clarify if the in vitro genotoxicity results are relevant in vivo, the next step would be to perform an in vivo genotoxicity testing as outlined in REACH Annex IX. Thus, a study proposal to perform an in vivo mammalian erythrocyte micronucleus test (OECD TG 474) was submitted.

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

Study period:

Dec. 2006 - Jan. 2007

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:

1997

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

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:

phenobarbital and ß-naphthoflavone induced rat liver S9-mix

Test concentrations with justification for top dose:

DOSE RANGE FINDING TEST: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
MUTATION ASSAY: 10, 33, 100, 333 and 1000 µg/plate

Precipitation of SAT 060283 on the plates was observed at the start and at the end of the incubation period at concentrations of 1000 µg/plate and upwards.

Vehicle / solvent:

The test substance was dissolved in dimethyl formamide (Merck, Darmstadt, Germany). Test substance concentrations were used within 3 hours after preparation.

Untreated negative controls:

other: solvent control served as negative control

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 2-nitrofluorene

Remarks:

without metabolic activation

Untreated negative controls:

other: solvent control served as negative control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

The characteristics of the different Salmonella typhimurium strains were as folIows:
- TA1537: hisC3076 Frameshift
- TA 98: hisD3052 / R-factor*, Frameshift
- TA 1535: hisG46, Base-pair substitutions
- TA 100: hisG46 / R-factor*, Base-pair substitutions
(*R-factor = plasmid pKM101 (increases error-prone DNA repair))

Each tester strain contained the following additional mutations:
rfa : deep rough (defective Iipopolysaccharide cellcoat)
gal : mutation in the galactose metabolism
chl : mutation in nitrate reductase
bio : defective biotin synthesis
uvrB : loss of the excision repair system (deletion of the ultraviolet-repair B gene)

The Salmonella typhimurium strains were regularly checked to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.

The Escherichia coli WP2uvrA strain detects base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of mutagens has been enhanced by permeabilization of the strain using Tris-EDTA treatment. The strain was regularly checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants.

Stock cultures of the five strains were stored in liquid nitrogen (-196°C).

Evaluation criteria:

A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The negative control data (number of spontaneous revertants per plate) should be within the laboratory historical range for each tester strain.
b) The positive control chemicals should produce responses in all tester strains, which are within the laboratory historical range documented for each positive contral substance. Furthermore, the mean plate count should be at least three times the concurrent vehicle control group mean.
c) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA 100 is not greater than two times the concurrent control, and the total number of revertants in tester strains TA 1535, TA 1537, TA98 or WP2uvrA is not greater than three times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA 100 is greater than two times the concurrent control, or the total number of revertants in tester strains TA 1535, TA1537, TA98 or WP2uvrA is greater than three times the concurrent control.
b) In case a positive response will be repeated, the positive response should be reproducible in at least one independently repeated experiment.

The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

No formal hypothesis testing was done.

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, T A98, TA 100

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 applicable

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 applicable

Positive controls validity:

valid

Dose range finding test

The test substance was tested in the tester strains TA 100 and WP2uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix.

- Precipitate: Precipitation of the test substance on the plates was observed at the start and at the end of the incubation period at concentrations of 1000 µg/plate and upwards.

- Toxicity: To determine the toxicity of the test substance, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined. No reduction of the bacterial background lawn was observed at any of the concentrations tested in both tester strains.

In strain TA 100 in the presence of S9-mix, fluctuations in the number of revertant colonies below the laboratory background historical control data range were observed. However, since no dose-relationship was observed, the reductions are not considered to be caused by toxicity of the test substance. In strain WP2uvrA, no biologically significant decrease in the number of revertants was observed.

- Mutagenicity: In the dose range finding test, no increase in the number of revertants was observed upon treatment with the test substance under all conditions tested.

Mutation assay

Based on the results of the dose range finding test, the test substance was tested up to concentrations of 1000 µg/plate in the absence and presence of 89-mix in two mutation assays.

The first mutation experiment was performed with the strains TA 1535, TA 1537 and TA 98 and the second mutation experiment was performed with the strains TA 1535, TA 1537, TA 98, TA 100 and WP2uvrA.

- Precipitate: Precipitation of the test substance on the plates was observed at the start and at the end of the incubation period at the concentration of 1000 µg/plate.

- Toxicity: In both mutation assays, there was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.

- Mutagenicity: In both mutation assays, no increase in the number of revertants was observed upon treatment with the test substance under all conditions tested.

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.

Based on the results of this study it is concluded that the est substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia eali reverse mutation assay.

Conclusions:

Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

Evaluation of the mutagenic activity of the test substance in the S. typhimurium reverse mutation assay and the E. coli reverse mutation assay (with independent repeat)

The test substance was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone) .

The study procedures described in this report were based on the most recent OECD and EEC guidelines.

The tesat substance was a clear yellowish liquid with a purity of 90%. The test substance was dissolved in dimethyl formamide.

In the dose range finding test, the test substance was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA 100 and WP2uvrA. The test substance precipitated on the plates at dose levels of 1000 µg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

Based on the results of the dose range finding test, the test substancewas tested in the first mutation assay at a concentration range of 10 to 1000 µg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA 1535, TA 1537 and TA98. In an independent repeat of the assay with additional parameters, the test substance was tested at the same concentration range as the first assay in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. SAT 060283 precipitated on the plates at the top dose of

1000 µg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

The test substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the

number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated

experiment.

In this study, 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.

Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation

assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2018 - November 2019

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:

in vitro mammalian chromosome aberration test

Target gene:

induction of chromosome breakage

Species / strain / cell type:

lymphocytes: Human

Details on mammalian cell type (if applicable):

Human peripheral blood lymphocytes from healthy and non-smoking donors with no known recent exposure to genotoxic chemicals and radiation

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix (phenobarbital and β-naphthoflavone induced)

Test concentrations with justification for top dose:

According to the relevant guidelines the highest recommended dose is 2 µL/mL. Due to the occurrence of precipitation of the test item the highest dose groups evaluated in the pre-experiment were 0.25 µL/mL (without metabolic activation) and 0.5 µL/mL (with metabolic activation).
Pre-Experiment: 0.005, 0.010, 0.025, 0.050, 0.10, 0.25, 0.50, 1.0, 1.5 and 2.0 µL/mL
Experiment I: without metabolic activation: 0.0025, 0.005, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5 µL/mL
Experiment I: with metabolic activation: 0.0025, 0.005, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1.0 µL/mL
Experiment II: without metabolic activation (not valid): 0.005, 0.01, 0.025, 0.05, 0.1, 0.25 µL/mL
Experiment II: without metabolic activation: 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5 µL/mL

Vehicle / solvent:

The test item was dissolved in THF (tetrahydrofurane; 0.5% v/v) and different stock solutions were prepared. The stock solutions were diluted with cell culture medium and added to the cells.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Human peripheral blood lymphocytes from healthy and non-smoking donors with no known recent exposure to genotoxic chemicals and radiation were used to examine the ability of chemicals to induce cytogenetic damage and thus to identify potential carcinogens or mutagens in vitro. For this study (in each experiment) blood was collected only from a single donor to reduce inter-individual variability.
Blood samples were drawn by venous puncture and collected in heparinized tubes. Before use the blood was stored under sterile conditions at 4 °C for a maximum of 4 h. Whole blood samples treated with an anti-coagulant (e. g. heparin) were pre-cultured in the presence of mitogen (phyto-haematogglutinin, PHA).

In the culture vessels 500 µL heparinized whole blood were added to 4.5 mL completed culture medium

Evaluation criteria:

EVALUATION
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
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 95% control limits of the historical negative control data.
When all of these criteria are met, the test chemical is then considered able to induce chromosomal aberrations in cultured mammalian cells in this test system.

Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined
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 95% control limits of the historical negative control data.
The test chemical is then considered unable to induce chromosomal aberrations in cultured human peripheral blood lymphocyte cells in this test system.

A test item is considered to be negative if there is no biologically relevant increase in the percentages of aberrant cells above concurrent control levels, at any dose group. Although most experiments will give clearly positive or negative results, in some cases the data set will preclude making a definitive judgement about the activity of the test substance.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

other: precipitation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

In experiment I, precipitate of the test item was noted at a concentration of 0.1 µL/mL as well as 0.25 µL/mL (withoutand withmetabolic activation) and higher concentrations.

In experiment II withoutmetabolic activation (not valid), no precipitation was observed in the tested concentrations of 0.005 µL/mL to 0.25 µL/mL. In experiment II (repetition) withoutmetabolic activation, precipitation occurred at a concentration of 0.1 µL/mL to 0.5 µL/mL.

In experiment I without metabolic activation, slight toxic effects (decrease below 70% rel. mitotic index) were seen at a concentration of 0.025 µL/mL, but were not regarded as relevant as no cytotoxic effects were noted at higher concentrations. In experiment I with metabolic activation, marginal toxic effects (decrease below 70% rel. mitotic index) were noted at concentrations of 0.01 and 0.025 µL/mL. Strong toxic effects were observed after treatment with higher concentrations (0.05 µL/mL – 0.25 µL/mL). In experiment II without metabolic activation, a biologically relevant decrease of the relative mitotic index was noted at concentrations of 0.01 to 0.25 µL/mL. The relative mitotic index was rather low and fluctuated between the first and second culture. Additionally, no concentration-dependency was observed with increasing concentrations.In experiment II (repetition) without metabolic activation, a biologically relevant decrease of the relative mitotic index was noted at a concentration of 0.05 µL/mL

The BrdU-technique was used for determining the proliferation index to detect a possible effect on the proliferation rate after treatment with the test item and thus indicating cell cycle delay. In the experiment I, the values of the proliferation index of the negative and solvent controls were 1.61 and 1.40 (without metabolic activation) as well as 1.38 and 1.31 (with metabolic activation). The proliferation index of the highest dose groups evaluated were 1.09 (0.1 µL/mL) (without activation) and 1.38 (0.25 µL/mL) (with metabolic activation).

In the experiment II (repetition), the values of the proliferation index of the negative and solvent controls were 1.70 and 1.33. The proliferation index of the highest concentration evaluated was 1.31 (0.05 µL/mL). A biologically relevant decrease of the proliferation index was indicated in experiment I without metabolic activation, which was not observed during the long-term treatment in experiment II.

There are several criteria for determining a positive result, such as a concentration-related increase or a reproducible increase in the number of cells with chromosome aberrationsfor at least one of the dose groups, which is higher than the laboratory negative control range.

In experiment I without metabolic activation, the aberration rate of the negative control (1.3%), the solvent control (1.7%) and all evaluated test item concentrations were within the historical control data of the testing facility (0.26 to 3.84% aberrant cells exclusive gaps,Table 16).With metabolic activation, the aberration rates of the negative control (1.0%), the solvent control (1.3%) and all dose groups treated with the test item were within the historical control data of the testing facility (-0.21 to 3.94% aberrant cells exclusive gaps,Table 16).The number of aberrant cells found in the dose groups treated with the test item did not show a biologically relevant increase compared to the corresponding negative control.

In experiment II without metabolic activation, the aberration rate of the negative control (2.0%) and the highest tested concentration of 0.25 µL/mL (2.3%) were within the historical control data of the testing facility (-0.33 to 3.60% aberrant cells exclusive gaps,Table 20). The number of aberrant cells found after test item treatment at lower concentrations and in the solvent control were increased compared to the corresponding negative control and the historic control range (solvent control: 4.0%; 0.025 µL/mL: 9.4%; 0.1 µL/mL: 7.6%). Moreover, less than 300 metaphases were present on the slides of the test item concentrations 0.025 µL/mL and 0.1 µL/mL. Due to the reduced evaluated metaphase number at low test item concentrations, the lack of a dose-dependent increase of chromosomal damage and the overall low and fluctuating mitotic index for the duplicate cultures, the experiment was classified as non-valid and had to be repeated.

In experiment II without metabolic activation, the aberration rate of the negative control (0.3%) and the solvent control (2.3%) were within the historical control data of the testing facility (-0.33 to 3.60% aberrant cells exclusive gaps,Table 16). The number of aberrant cells found after test item treatment at different concentrations was increased compared to the corresponding negative control and the historic control range (0.01 µL/mL: 4.3%; .0.02 µL/mL: 4.7% and 0.05 µL/mL: 5.3%).

The Fisher´s exact test was performed to verify the results in the experiment. No statistically significant increase (p < 0.05) of cells with chromosomal aberrations was noted in the dose groups of the test item evaluated in experiment I without and with metabolic activation. In experiment II (not valid) without metabolic activation a statistically significant increase was determined at 0.025 µL/mL, but not at higher evaluated concentrations. In the repetition experiment II, no statistical significance was observed in any of the analysed concentrations.

The Chi-squared test for trend was performed to test whether there is a concentration-related increase in chromosomal aberrations. No statistically significant increase was observed in experiment I without and with metabolic activationand in experiment II (repetition) without metabolic activation. In the experiment II (not valid) a statistical significance was noted but not considered for further evaluation since a decrease was detected.

EMS (400 and 600 µg/mL) and CPA (5 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations, thus proving the ability of the test system to indicate potential clastogenic effects.

Conclusions:

In conclusion, it can be stated that during the described in vitro chromosomal aberration test and under the experimental conditions reported, the test item Sa 34 did induce structural chromosomal aberrations in human lymphocyte cells detected after the long-term treatment.
Therefore, Sa 34 is considered to be clastogenic in this chromosome aberration test.

Executive summary:

A chromosome aberration assay was carried out in order to investigate a possible potential of Sa 34 to induce structural chromosome aberrations in human lymphocytes.

The metaphases were prepared 24 h after start of treatment with the test item. The treatment interval was 4 h without and with metabolic activation (experiment I) and 24 h without metabolic activation (experiment II). Duplicate cultures were set up. Per culture 150 metaphases were scored for structural chromosomal aberrations.

The following concentrations were evaluated:

Experiment I:

Without metabolic activation, 4 h treatment, 24 h preparation interval:

           0.025, 0.050 and 0.100 µg/mL

With metabolic activation, 4 h treatment, 24 h preparation interval:

           0.05, 0.10 and 0.25 µg/mL

Experiment II

Without metabolic activation(not valid), 24 h treatment, 24h preparation interval:

           0.025, 0.1 and 0.25 µL/mL

Without metabolic activation, 24 h treatment, 24 h preparation interval:

           0.01, 0.02 and 0.05 µg/mL

 

In experiment I, precipitate of the test item was noted at a concentration of 0.1 µL/mL as well as 0.25 µL/mL (without and with metabolic activation) and higher concentrations. In experiment II without metabolic activation, precipitation occurred at a concentration of 0.1 µL/mL to 0.5 µL/mL.

In experiment I without metabolic activation, slight toxic effects (decrease below 70% rel. mitotic index) were seen at a concentration of 0.025 µL/mL, but were not regarded as relevant as no cytotoxic effects were noted at higher concentrations. In experiment I with metabolic activation, marginal toxic effects (decrease below 70% rel. mitotic index) were noted at concentrations of 0.01 and 0.025 µL/mL. Strong toxic effects were observed after treatment with higher concentrations (0.05 µL/mL – 0.25 µL/mL).

In experiment I without metabolic activation, a biologically relevant decrease of the proliferation index was observed. After the long-term treatment, the proliferation index was not reduced.

In experiment II without metabolic activation (not valid), a biologically relevant decrease of the relative mitotic index was noted at concentrations of 0.01 to 0.25 µL/mL. In the repetition experiment II, cytotoxic effects occurred already at lower concentrations of 0.05 µL/mL.

No increase in the frequencies of polyploid cells was found after in all experimental conditions.

In experiment I, no biologically relevant increase of the aberration rates was noted after treatment with the test item without and with metabolic activation. The aberration rates of all dose groups treated with the test item were within the historical control data of the solvent control.

In experiment II without metabolic activation, the aberration rate of the solvent control and after test item treatment at 0.025 µL/mL and 0.1 µL/mL were increased compared to the corresponding negative control and the historic control range. Moreover, less than 300 metaphases were present on the slides of these concentrations. In contrast to these results, no chromosomal damage was observed at the highest tested concentration. Due to the reduced evaluated metaphase number at low test item concentrations, the lack of a dose-dependent increase of chromosomal damage and the overall low and fluctuating mitotic index for the duplicate cultures, the experiment was classified as non-valid and had to be repeated.

In experiment II without metabolic activation (repetition) an increase of aberrant cells was noted at concentrations of 0.01 µL/mL, 0.02 µL/mL and 0.05 µL/mL. This increase was considered as biologically relevant since the increase was above the historic control range.

The Fisher´s exact test was performed to verify the results in the experiment. No statistically significant increase (p < 0.05) of cells with chromosomal aberrations was noted in the dose groups of the test item evaluated in experiment I without and with metabolic activation. In experiment II (not valid) without metabolic activation a statistically significant increase was determined at 0.025 µL/mL, but not at higher evaluated concentrations. In the repetition experiment II, no statistical significance was observed in any of the analysed concentrations.

The Chi-squared Test for trend was performed to test whether there is a concentration-related increase in chromosomal aberrations. No statistically significant increase was observed in experiment I without and with metabolic activationand in experiment II (repetition) without metabolic activation. In the experiment II (not valid) a statistical significance was noted but not considered for further evaluation since a decrease was detected.

EMS (400 and 600 µg/mL) and CPA (5 µg/mL) were used as positive controls and induced distinct and biologically relevant increases of chromosomal aberrations, thus proving the efficiency of the test system to indicate potential clastogenic effects.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2018 - March 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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT-gene for hypoxanthin-guanin-phosphoribosyl-transferase

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix (phenobarbital and β-naphthoflavone induced)

Test concentrations with justification for top dose:

Pre-Experiment: with and without metabolic activation
0.010, 0.025, 0.050, 0.1, 0.25, 0.5, 1.0 and 2.0 μL/mL
Main-Experiment:
without metabolic activation
0.10, 0.12, 0.14, 0.16, 0.18, 0.20, 0.25, 0.50, 1.0 and 2.0 μL/mL
with metabolic activation
0.010, 0.025, 0.050, 0.10, 0.25, 0.50, 1.0 and 2.0 μL/mL

Vehicle / solvent:

The test item was dissolved in THF (tetrahydrofurane; 0.5% v/v), processed by ultrasound for 15 min at 37 °C and different stock solutions were prepared. The stock solutions were diluted with cell culture medium and added to the cells.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

V79 cells in vitro have been widely used to examine the ability of chemicals to induce cytogenetic changes and thus identify potential carcinogens or mutagens. These cells are characterized by their high proliferation rate (12 - 14 h doubling time of the Eurofins BioPharma Product Testing Munich GmbH stock cultures) and their high cloning efficiency of untreated cells (usually more than 50%). These features of the cells are necessary for the appropriate performance of the study.

The V79 cells (ATCC, CCL-93) were stored over liquid nitrogen (vapour phase) in the cell bank of Eurofins BioPharma Product Testing Munich GmbH. This allows the repeated use of the same cell culture batch in experiments. Each cell batch was routinely checked for mycoplasma infections (via PCR), stable spontaneous mutant frequency as well as stability of the modal chromosome number. Freshly thawed cells from stock cultures were maintained in plastic culture flasks in minimal essential medium (MEM) and cultured at a humidified atmosphere of 5% CO2 and at 37 °C incubation temperature.

For purifying the cell population of pre-existing HPRT- mutants cells were exposed to HAT medium containing 10 μM hypoxanthine, 3.2 μM aminopterin, 5 μM thymidine and 10 μM glycine for several cell doublings (2 - 3 days) with a subsequent recovery period in medium supplemented with 10 μM hypoxanthine and 5 μM thymidine.

Pre-Experiment and Main-Experimtent were performed as a 4 h short-term exposure assay.

Evaluation criteria:

ACCEPTABILITY
A mutation assay is considered acceptable if it meets the following criteria:
- Negative and/or solvent controls fall within the performing Iaborateries historical control data range: 1-39 mutants/10E6 cells;
- The absolute cloning efficiency: ([number of positive cultures x 100) / total number of seeded cultures) of the negative and/or solvent controls is > 50%
- The positive controls (EMS and DMBA) induce significant increases (at least 3-fold increase of mutant frequencies related to the comparable negative control values and higher than the historical range ofnegative controls) in the mutant frequencies.

EVALUATION
A test is considered to be negative if there is no biological relevant increase in the number of mutants.
There are several criteria for determining a positive result:
- a reproducible three times higher mutation frequency than the solvent control for at least one of the concentrations;
- a concentration related increase of the mutation frequency; such an evaluation may be considered also in the case that a three-fold increase of the mutant frequency is not observed;
- if there is by chance a low spontaneaus mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.
According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

In the main experiment 0.50 μL/mL (without metabolic activation) and 1.0 μL/mL (with metabolic activation) were selected as the highest concentrations. The experiments with and without metabolic activation were performed as a 4 h short-term exposure assay. The pH-value detected with the test item and the osmolality were within the physiological range.

The test item was investigated at the following concentrations:

without metabolic activation:

0.16, 0.18, 0.20, 0.25 and 0.50 μL/mL

and with metabolic activation:

0.05, 0.10, 0.25, 0.50 and 1.0 μL/mL

In main experiment without metabolic activation precipitation was observed at concentration of 0.50 μL/mL (and higher) and in the experiment with metabolic activation at concentrations of 1.0 μL/mL (and higher).

No biologically relevant growth inhibition (reduction of relative survival below 70%) was observed after the treatment with the test item in the experiment with and without metabolic activation.

In the main Experiment without and with metabolic activation all validity criteria were met. The mutant values of the negative and solvent controls fall within the historical data range of the test facility and the cloning efficiencies of the negative and solvent controls are > 50%.

The positive controls, DMBA (1.0 μg/mL) and EMS (300 μg/mL) showed statistically significant increases in mutant frequency, thereby demonstrating both the sensitivity and validity of the test systems.

Experiment without metabolic activation

In the experiment without metabolic activation the mutant values of the negative, solvent controls and all mutant values of the test item concentrations found were within the historical control data of the test facility Eurofins Munich (about 8.5 - 40.2 mutants per 106 cells). The positive control EMS induced a distinct increase in mutant frequency with 261.1 mutants/106 cells.

The mutant frequencies induced by the test item did not show a biologically relevant increase. None of the observed mutant frequencies was statistically significantly increased over those of the solvent controls.

The highest mutant frequency was observed at a concentration of 0.5 μL/mL (34.2 mutants per 106 cells) with a relative survival of 124%.

In the experiment with metabolic activation the mutant values of the negative, solvent controls and most mutant values of the test item concentrations found were within the historical control data of the test facility Eurofins Munich (about 9.6 - 44.0 mutants per 106 cells). The positive control DMBA induced a distinct increase in mutant frequency with 329.2 mutants/106 cells.

The mutant frequencies induced by the test item did not show a biologically relevant increase. None of the observed mutant frequencies was statistically significantly increased over those of the negative controls. Although for the highest tested concentration (1.0 μL/mL) the mutant frequency was above the historical control data (mutant frequency with 45.5 mutants/106 cells), this increase was considered as not biologically relevant, since this was only a slight increase and no statistically relevant increase and no concentration-response relationship was evaluated. Additionally, precipitation was observed at this concentration therefore the increased mutant frequency has to be evaluated with caution, as artefactual effects may result from the precipitate.

Conclusions:

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item Sa 34 is considered to be non-mutagenic at the HPRT locus using V79 cells of the Chinese Hamster.

Executive summary:

The test item Sa 34 was assessed for its potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster.

The selection of the concentrations was based on data from the pre-experiments. The experiments with and without metabolic activation were performed as a 4 h short-term exposure assay.

The test item was investigated at the following concentrations:

without metabolic activation:

0.16, 0.18, 0.20, 0.25 and 0.50 μL/mL

and with metabolic activation:

0.05, 0.10, 0.25, 0.50 and 1.0 μL/mL

Precipitation of the test item was noted in pre-experiment (without and with metabolic activation) at concentrations 1.0 μL/mL and higher.

In main experiment without metabolic activation precipitation was observed at concentration of 0.50 μL/mL (and higher) and in the experiment with metabolic activation at concentrations of 1.0 μL/mL (and higher).

No growth inhibition was observed in the experiment without and with metabolic activation.

In the experiments no biologically relevant increase of mutants was found after treatment with the test item (without and with metabolic activation). All mutant values are within the historical data base of the test facility.

A statistical analysis displayed that one of the mutant frequency of the experiment with metabolic activation was significantly increased over those of the solvent controls, but no dose-response relationship was determined in the χ² test for trend. Additionally, the mutant frequency for this concentration was above the historical control data. But due to the occurrence of precipitation and this was only a slight increase, this effect was considered as not biologically relevant.

No dose-response relationship was observed.

DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

REACH_negative | rat (male/female) | OECD 474 | #key study#

In order to clarify the genotoxic potential, an in vivo combined micronucleus assay was conducted according to OECD TG 474. In the respective micronucleus study the test substance was not clastogenic or aneugenic up to a dose of 2000 mg/kg bw under the experimental conditions. In conclusion, the test item is not considered to have a genotoxic potential in vivo. 

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September - December 2021

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:

20 July, 2016

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

August 1998

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

February 14, 2017

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

ANIMALS

Species: Rat (mus musculus)
Strain: Healthy Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Number of animals: 5 of each sex per dose group (7 animals for 1 MTD)
Initial age at start of acclimatisation: 6 - 8 weeks
Age at start of treatment: Minimum 7 weeks
The animals were derived from a controlled full barrier maintained breeding system (spf). According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals were bred for experimental purposes and underwent an adequate acclimatisation period after arrival (at least five days). The animals were randomly distributed to test groups.

HUSBANDRY

The animals were barrier maintained (semi-barrier) in an air conditioned room. The experiment was conducted under standard laboratory conditions.
Housing: 5 animals of identical sex per cage
Cage type: IVC cage (Polysulphone), Type III H
Bedding: Altromin saw fiber bedding (Batch: 1238)
Feed: Free access to Altromin 1324 (Batch: 1033) maintenance diet for rats and mice
Air change: At least 10 x per hour
Water: Free access to tap water, sulphur acidified to pH value of approx. 2.8 (drinking water, municipal residue control, micro-biologically controlled at frequent intervals)
Environment: Temperature 22 ± 3 °C
Relative humidity 55 ± 10%
Artificial light 6:00 - 18:00
Certificates of food, water and bedding was filed for two years at BSL Munich and afterwards archived at Eurofins Munich.
The animals were randomly distributed into the test groups. The animals of each test group and sex were housed in separate cages that were individually marked (study number, sex, control/test group). The animals of each cage were individually marked for identification by tail and ear drawing.

Route of administration:

intraperitoneal

Vehicle:

corn oil

Details on exposure:

The test item formulations were prepared freshly one hour prior to administration at the Test Facility. The prepared formulations were transferred to the Test Facility protected from light together with the control formulations under consideration of stability.
The test item was diluted in Corn oil. All animals received a single volume intraperitoneally of 10 mL/kg bw. The solvent was chosen according to its relative non-toxicity for the animals.

Duration of treatment / exposure:

single administration

Frequency of treatment:

single administration

Post exposure period:

48 and 72 h

Dose / conc.:

400 mg/kg bw (total dose)

Dose / conc.:

1 000 mg/kg bw (total dose)

Dose / conc.:

2 000 mg/kg bw (total dose)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Name: CPA; Cyclophosphamide
CAS No.: 50-18-0
Supplier: Sigma
Catalogue No.: C0768
Batch No.: MKCL2547
Dissolved in: physiological saline
Dosing: 10 mg/kg bw
Route and frequency of administration: ip, single
Volume administered: 10 mL/kg bw
Expiry date: October 2022
The solution was aliquoted and stored at <=-15°C. On the day of administration, the solution was freshly thawed. The stability of CPA at room temperature is quite good (3.5% is hydrolysed per day in aqueous solution). It is acceptable that the positive control can be administered by a route different from the test agent and sampled only at a single time. The sampling time for the positive control was 48 h after treatment

Tissues and cell types examined:

Peripheral blood - Erythrocytes

Details of tissue and slide preparation:

Blood cells were immediately fixed in ultracold methanol. For the analysis (at the earliest 72 h after fixation) the MicroFlow Kit (Litron Laboratories, Rochester, New York, USA) was used and the procedures described in the manual were followed for analyzing peripheral blood samples for the presence of micronuclei. Briefly, blood cells were washed in Hank’s Balanced Salt Solution, centrifuged at 400 x g for 5 minutes at 4 °C and the supernatant was discarded. Blood cell populations were discriminated using specific antibodies against CD71 (expressed only at the surface of immature erythrocytes) and CD61 (expressed at the surface of platelets) and DNA content of micronuclei, was determined by the use of a DNA specific stain (propidium iodide, PI). Evaluation of all samples, including those of positive (CPA) and negative controls, were performed using a flow cytometer (FACSLyric, BD Biosciences). Anti-CD71 antibodies were labeled with fluorescein isothiocyanate (FITC), anti-CD61 antibodies were labelled with phycoerythrin (PE). Particles were differentiated using forward scatter (FSC) and side scatter (SSC) parameters of the flow cytometer. Fluorescence intensities were recorded on PMT position E, D and B for FITC, PE and PI, respectively. 10000 immature erythrocytes per animal were scored for the incidence of micronucleated immature erythrocytes. To detect a possibly occurring cytotoxic effect of the test item, the ratio between immature and mature erythrocytes was determined. The result was expressed as relative PCE (rel. PCE = proportion of polychromatic (immature) erythrocytes among total erythrocytes).

Statistics:

Statistical methods applied used the animal as the experimental unit.
Pairwise comparison of the proportion of PCE among total erythrocytes as well as the proportion of micronucleated polychromatic (immature) erythrocytes (PCE) among total PCE between the control groups and each of the treatment groups was performed by means of the non-parametric Mann-Whitney test at a statistical significance level of 5% (p < 0.05, two-tailed).
The y² test for trend at a statistical significance level of 5% (p < 0.05, two-tailed) was used to test whether there is a dose-related increase in the micronucleated cells frequency of the dose groups of the 48 h sampling time.
Statistical methods were performed using the software GraphPad Prism version 6.0.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

other: vehicle control served as negative control

Positive controls validity:

valid

Pre-Experiment

The test item was diluted in Corn oil within 1 h before treatment. 200 mg/mL of the test item was used as highest dose group corresponding to 2000 mg/kg bw according to the OECD guideline 474 [5].

The selection of the highest dose was conducted in accordance with the following current international guidelines for assessment of acute toxicity (OECD 423 and OECD 425), particularly with respect to selection of dose spacing and animal welfare aspects.

In the pre-experiment a concentration of 200 mg/mL of the test item was evaluated. Three male and three female rats received a single dose of 2000 mg/kg bw ip and showed toxicity such as reduction of spontaneous activity, prone position, piloerection, ataxia, opisthotonos, diarrhoea, abnormal breathing and half eyelid closure. In male rats the clinical signs persisted up to 48 h after application. The female rats were fully recovered 4 hours after application (Table 1).

Due to the results obtained in the pre-experiment 2000 mg/kg bw was chosen as maximum tolerated dose (1 MTD) in the main experiment.

Signs of Toxicity observed in the Pre-Experiment (2000 mg/kg bw)

Signs of Toxicity	Time post-application / Sex (3 male and 3 female rats)
	30 min		1 h		2 h		4 h		24 h		48 h		72 h
	m	f	m	f	m	f	m	f	m	f	m	f	m	f
Reduction of spontaneous activity	3	3	3	2	3	0	2	1	2	0	2	0	0	0
Prone position	1	1	0	0	0	0	0	0	0	0	0	0	0	0
Piloerection	0	0	0	0	2	0	2	0	0	0	2	0	0	0
Ataxia	0	2	0	1	1	0	0	0	0	0	0	0	0	0
Opisthotonos	1	1	0	0	0	0	0	0	0	0	0	0	0	0
Diarrhoea	1	0	2	1	2	1	2	1	0	0	0	0	0	0
Abnormal breathing	0	0	0	1	0	0	0	0	0	0	0	0	0	0
Half eyelid closure	0	0	1	0	0	0	0	0	0	0	0	0	0	0

m:     male
f:       female

Toxicity in the Main Experiment

2000 mg/kg bw was tested as the maximum tolerated dose (1 MTD) in the main experiment. The volume administered i.p. was 10 mL/kg bw.

Animals treated with the highest dose (1 MTD) showed moderate toxic effects (Table 2) with reduction of spontaneous activity, prone position, piloerection, ataxia, hunched posture, constricted abdomen, opisthotonos, abnormal breathing and half eyelid closure up to 4 h after application. After 24 h only reduction of spontaneous activity and piloerection were still seen and 48 h after application only reduction of spontaneous activity was still observed for two male animals.

Rats treated with 1000 mg/kg bw (0.5 MTD) showed the same clinical signs as the 1 MTD animals except for abnormal breathing and the clinical signs were less pronounced and the animals recovered faster.

The male animals treated with 400 mg/kg bw (0.2 MTD) showed mild toxic effects after the treatment with the test item up to 4 h after application. Female animals treated with 400 mg/kg bw (0.2 MTD) did not show any clinical signs

Signs of Toxicity observed in the Main Experiment (2000 mg/kg bw)

Signs of Toxicity	Time post-application / Sex (5 male and 5 female rats)
	30 min		1 h		2 h		4 h		24 h		48 h		72 h
	m	f	m	f	m	f	m	f	m	f	m	f	m	f
Reduction of spontaneous activity	5	5	5	5	5	5	5	5	4	1	2	0	0	0
Prone position	5	5	5	2	4	5	0	0	0	0	0	0	0	0
Piloerection	0	0	1	4	1	4	5	4	3	1	0	0	0	0
Ataxia	5	1	0	2	1	2	0	0	0	0	0	0	0	0
Hunched posture	0	0	0	3	0	0	5	5	0	0	0	0	0	0
Constricted abdomen	0	0	2	3	4	2	5	2	0	0	0	0	0	0
Opisthotonos	0	0	3	0	2	0	0	0	0	0	0	0	0	0
Abnormal breathing	0	0	1	1	0	0	0	1	0	0	0	0	0	0
Half eyelid closure	5	4	5	5	4	5	4	4	0	0	0	0	0	0

m:     male
f:       female

Relative PCE

The relative PCE (rel. PCE = proportion of polychromatic (immature) erythrocytes among total erythrocytes) was determined for each animal. The relative PCE is the supportive endpoint to assess cytotoxicity, which helps to demonstrate a target cell exposure with the test item.

The negative controls (48 h, 72 h) were within the historical control limits of the negative control (0.64% - 2.46% for males, 0.30% - 1.81% for females). The mean values noted for the 48 h negative control were 1.14% for males and 1.29 for females. For the 72 h negative control mean values of 1.24% for males and 1.34% for females were observed.

The animal group treated with 0.2 MTD showed mean values of the relative PCE of 1.32% for males and 0.72% for females. The mean value observed in the male group was within the range of the concurrent negative control and within the historical control limits of the negative control. The mean value observed for the female group was statistically significantly decreased compared to the concurrent negative control but was still within the historical control limits.

The dose group, which was treated with 0.5 MTD, showed mean values of the relative PCE of 0.82% for males and 0.67% for females. The mean value observed in the male group was within the range of the concurrent negative control and within the historical control limits of the negative control. The mean value observed for the female group was statistically significantly decreased compared to the concurrent negative control but was still within the historical control limits.

The animals who received 1 MTD (48 h sampling) showed mean values of the relative PCE of 0.71% for males and 0.36% for females. The mean value observed in the male group was within the range of the concurrent negative control and within the historical control limits of the negative control. The mean value observed for the female group was statistically significantly decreased compared to the concurrent negative control but was still within the historical control limits.

The animal group which was treated with 1 MTD (72 h sampling) showed mean values of the relative PCE of 1.09% for males and 0.84% for females (Table 10). The mean values observed in the male and female group were within the range of the concurrent negative control and within the historical control limits of the negative control. (Figure 2).

The decrease of PCE values in treated animals compared to control animals is related to toxicity and therefore also a hint for a target cell exposure of the test item.

Micronucleated polychromatic erythrocytes

For all dose groups, including positive and negative controls, 10000 immature erythrocytes per animal were scored for the incidence of micronucleated immature erythrocytes.

The negative controls (48 h and 72 h) evaluated were within the historical control limits of the negative control (0.03 – 0.15% for males, 0.03 – 0.12% for females). The mean values of micronuclei observed for the negative control were 0.08% for males and 0.04% for females after 48 h and 0.09% for males and 0.09% for females after 72 h.

The mean values of micronuclei observed after treatment with 0.2 MTD were 0.08% for males and 0.07% for females. The mean value observed in the male group was within the range of the concurrent negative control as well as within the historical control limits of the negative control. The mean value observed for the female group was slightly increased compared to the concurrent negative control but this was not statistically significant and the value was within the historical control limits.

The mean values noted for the 0.5 MTD dose groups were 0.10% for males and 0.10% for females. The mean value observed in the male group was within the range of the concurrent negative control as well as within the historical control limits of the negative control. The mean value observed for the female group was statistically significantly increased compared to the concurrent negative control. Since the value was within the historical control limits and no dose-response relationship was observed, this increase was considered as not biologically relevant.

The dose group treated with 1 MTD (48 h sampling) showed mean values of 0.09% for males and 0.05% for females. The mean values observed in the male and female group were within the range of the concurrent negative control as well as within the historical control limits of the negative control.

The mean values observed for the 1 MTD (72 h sampling) were 0.09% for males and 0.07% for females. The mean values observed in the male and female group were within the range of the concurrent negative control as well as within the historical control limits of the negative control.

No biologically relevant increase of micronuclei was found after treatment with the test item in any of the dose groups evaluated.

The nonparametric Mann-Whitney Test was performed to verify the results. No statistically significant increases (p< 0.05) of cells with micronuclei were noted in the dose groups of the test item evaluated except for the female 0.5 MTD dose group that showed a statistically significant increase compared to the concurrent negative control. However, the value was within the historical control limits of the negative control and no dose-response relationship was observed. Based on these data this increase was regarded as not biologically relevant.

Additionally, the c² Test for trend was performed to test whether there is a dose-related increase in the micronucleated cells frequency of the dose groups of the 48 h sampling time. No statistically significant increase in the frequency of micronucleated cells was observed.

Cyclophosphamide (10 mg/kg bw) administered ip was used as positive control, which induced a statistically significant increase in the micronucleus frequency (mean percentage of cells with micronuclei was 0.72% for male and 1.05% for female rats (Table 5)). This demonstrates the validity of the assay.

 

 

 

 

Conclusions:

In conclusion, it can be stated that during the study and under the experimental conditions reported, the test item Sa 34 did not induce structural and/or numerical chromosomal damage in the immature erythrocytes of the rat.
Therefore, the test item Sa 34 is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the Mammalian Erythrocyte Micronucleus Test.

Executive summary:

This study was performed to investigate the potential of Sa 34 to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the rat. After expulsion of the main nucleus, PCE are released into peripheral blood. Peripheral blood was sampled and analysed for presence of micronucleated PCE

The test item was diluted in Corn oil. The volume administered ip was 10 mL/kg bw. Peripheral blood samples were collected for micronuclei analysis 48 h and 72 h after a single application of the test item.

A pre-experiment was performed as dose range finding study based on the OECD guideline 474 and other relevant documents (OECD 423 and OECD 425). Based on the outcome of the dose range finding study, a dose of 2000 mg/kg bw was selected as maximum tolerated dose (MTD).

In the main experiment three dose levels were used covering a range from the maximum tolerated dose to little or no toxicity. The following dose groups were selected based on the toxicity observed in the pre-experiment:

Dose Group	Dose [mg/kg bw]
0.2 MTD	400
0.5 MTD	1000
1 MTD	2000

 

No toxicity was observed in female animals during the treatment with 0.2 MTD. Male animals showed mild symptoms after treatment with 0.2 MTD. Animals treated with 0.5 MTD showed mild signs of systemic toxicity. The animals treated with a dose of 1 MTD showed moderate signs of systemic toxicity such as with reduction of spontaneous activity, prone position, piloerection, ataxia, hunched posture, constricted abdomen, opisthotonos, abnormal breathing and half eyelid closure.

For all dose groups, including positive and negative controls, 10000 polychromatic erythrocytes per animal were scored for incidence of micronucleated immature erythrocytes. The negative controls (48 h, 72 h) were within the historical control limits of the negative control. The mean values noted for the dose groups which were treated with the test item were within the range of the concurrent negative control except for the female 0.5 MTD dose group that showed a statistically significant increase. Since the value was within the historical control limits and no dose-response relationship was observed, this increase was considered as not biologically relevant.

No biologically relevant increase of micronuclei was found after treatment with the test item in any of the dose groups evaluated.

The nonparametric Mann-Whitney Test was performed to verify the results. No statistically significant increases (p< 0.05) of cells with micronuclei were noted in the dose groups of the test item evaluated except for the female 0.5 MTD dose group that showed a statistically significant increase compared to the concurrent negative control. However, the value was within the historical control limits of the negative control and no dose-response relationship was observed. Based on these data this increase was regarded as not biologically relevant.

Additionally, the c² Test for trend was performed to test whether there is a dose-related increase in the micronucleated cells frequency of the dose groups of the 48 h sampling time. No statistically significant increase in the frequency of micronucleated cells was observed.

Cyclophosphamide (10 mg/kg bw) administered ip was used as positive control, which induced a statistically significant increase in the micronucleus frequency. This demonstrates the validity of the assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The substance was clearly negative in the in vivo micronucleus assay conducted according to OECD TG 474 and in the in vitro gene mutation assays OECD TG 471 and OECD TG 476, thus the classification criteria are not met.