Amidation products of C16-18 (even numbered), C18 unsaturated fatty acids esters with 1,1'-iminodipropan-2-ol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic Toxicity In Vitro

Ames Assay (Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay)

Not mutagenic.

Chromosome Aberration (in cultured peripheral human lymphocytes)

MLA-3202 is not clastogenic in human lymphocytes.

Mammalian Cell Gene Mutation (mouse lymphoma L5178Y)

Not mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Micronucleus test

In conclusion, MLA-3202 (Naugalube OFM 3202) is not clastogenic or aneugenic in the bone marrow micronucleus test of male mice up to a dose of 2000 mg/kg (the maximum recommended dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

15 Sep 2017 to 02 Nov 2017.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

GLP study in accordance with current guidelines.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

OECD Guideline 474. Mammalian Erythrocyte Micronucleus Test, (adopted 29 July 2016).

Deviations:

yes

Remarks:

See "Any other information" for details

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

No further details specified in the study report.

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

This type of study plan was reviewed and agreed by the Laboratory Animal Welfare Officer and the Ethical Committee of Charles River Den Bosch as required by the Dutch Act on Animal Experimentation (February 1997).
NMRI mice (SPF) were used as the test system. These mice are recommended by international guidelines (e.g. OECD, EC). Females were nulliparous and non-pregnant. The animals were provided by Charles River, Sulzfeld, Germany.

Sex:

male

Details on test animals or test system and environmental conditions:

Young adult animals were selected (6 weeks old at the start of treatment). The total number of animals used in the dose-range finding study was 6 and in the main study 55. In the micronucleus main study 5 male mice were treated per sampling time in each treatment group. In addition 15 male mice were treated with the highest dose group and vehicle control group for blood sampling.
The body weights of the mice at the start of the treatment in the main study were within 20% of the sex mean. The mean body weights were 33.6 ± 2.0 g and the range was 29 - 38 g. The mice were identified by a unique number on the tail written with a marker pen. The animals were allocated at random to the treatment groups.
The acclimatization period was at least 5 days before the start of treatment under laboratory conditions.
On arrival and at the start of the treatment, all animals were clinically examined to ensure selected animals were in a good state of health.

Husbandry
Room number
The animals were housed in room number A.018.

Environmental Conditions
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 22°C with an actual daily mean relative humidity of 47 to 61%. A 12-hour light/12-hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Accommodation
The animals were group housed (maximum 5 animals per sex per cage) in labelled Macrolon cages (type MIII height 180 mm, length 380 mm and width 220 mm) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany). Paper bedding (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom) was provided as cage-enrichment.

Diet
The animals had free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). Results of analyses for nutrients and contaminants of each batch were examined and archived.

Water
The animals had free access to tap-water. Certificates of analysis (performed quarterly) were examined and archived.
Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

Route of administration:

oral: gavage

Vehicle:

The vehicle of the test item was milli-Q water.

Details on exposure:

Dose-range Finding Study
Selection of an adequate dose-range for the micronucleus main test was based on a dose-range finding study. The test procedure and conditions were similar to those applied in the main test.
One dose group, comprising of 3 males and 3 females, was dosed with 2000 mg/kg body weight, the highest concentration that was used for the main study. The observation period after dosing was three days. During this period mortality and physical condition were recorded at least once a day.

Micronucleus Main Test
Based on the results of the dose-range finding test a full study with one sex was performed. Since there were no differences in toxicity between sexes, only male animals were used in the main study.
The test item showed no toxicity in the dose-range finding study up to 2000 mg/kg body weight, the highest dose required in the guidelines. Therefore, 2000 mg/kg was selected as the dose to be tested.
At least five male mice were used per sampling time in each treatment group. The animals were dosed twice with a 24 hours interval and sampled at 48 hours.

No correction was made for the purity/composition of the test item.
A solubility test was performed based on visual assessment. MLA-3202 (Naugalube OFM 3202) was suspended in water. MLA-3202 (Naugalube OFM 3202) concentrations were treated with ultra-sonic waves to obtain a homogeneous suspension. MLA-3202 (Naugalube OFM 3202) concentrations were dosed within 2.5 hours after preparation.
Any residual volumes were discarded.

Duration of treatment / exposure:

The animals were dosed twice with a 24 hours interval

Frequency of treatment:

In the main study male animals were dosed twice via oral gavage with vehicle or with 2000, 1000 and 500 mg MLA-3202 (Naugalube OFM 3202) per kg body weight. A positive control group was dosed once via oral gavage with 40 mg cyclophosphamide (CP) per kg body weight.

Post exposure period:

48 hours

Dose / conc.:

500 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

Dose / conc.:

2 000 mg/kg bw/day

No. of animals per sex per dose:

At least five male mice were used per sampling time in each treatment group.

Control animals:

yes, concurrent vehicle

Positive control(s):

The positive control used in the micronucleus test was cyclophosphamide (CP; CAS No. 50-18-0; Baxter B.V., Utrecht, The Netherlands) dissolved in physiological saline (Eurovet Animal Health, Bladel, the Netherlands) dosed as a single oral intubation of 40 mg/kg body weight.
The route of administration and the volume administered of the positive control were the same as those of the test item.

Tissues and cell types examined:

The micronucleus test is a mammalian in vivo cytogenetic test, which detects damage to the chromosome or to the mitotic apparatus induced by a test item.
Basis for this test is the increase in the number of micronucleated polychromatic erythrocytes in the bone marrow of the femur of mice exposed to the test item compared with control animals. Micronuclei are small particles consisting of acentric fragments of chromosomes or entire chromosomes, which lag behind at anaphase stage of cell division. After telophase, these fragments may not be included in the nuclei of daughter cells and form single or multiple micronuclei in the cytoplasm. When an erythroblast develops into an erythrocyte, the main nucleus is extruded and may leave micronuclei in the cytoplasm. Visualization of micronuclei is facilitated in polychromatic cells because they lack a nucleus. Polychromatic erythrocytes can be distinguished from normochromatic cells by their bluish color after staining.

Details of tissue and slide preparation:

Blood Sampling and Plasma Preparation
Blood was collected from 3 satellite animals of the highest dose group and vehicle control group per time point 1, 2, 4, 6 and 8 hours after second dosing. No blood was collected from the positive control group (CP). Blood samples were collected under iso-flurane (Abbott Laboratories Ltd., Zwolle, The Netherlands) anesthesia from the heart/vena cava (terminal procedure). In addition, blood was collected from the animals of group A and B just before bone marrow sampling. Samples were collected under iso-flurane anesthesia from the heart/vena cava (terminal procedure).
Approximately 300 μl blood samples per animal were taken and collected into tubes prepared with K2-EDTA.
Within two hours after blood sampling, plasma from all samples was harvested by centrifugation for 10 minutes at 3220 g at 5°C.
Plasma was stored in labelled polypropylene tubes at < - 75°C until dispatch.

Isolation of Bone Marrow
Bone marrow was sampled 48 hours after the first dosing. The animals were sacrificed by cervical dislocation. Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 2 ml of fetal calf serum (Invitrogen Corporation, Breda, The Netherlands). The cell suspension was collected and centrifuged at 216 g for 5 min.

Preparation of Bone Marrow Smears
The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet.
The cells in the sediment were carefully mixed with the remaining serum. A drop of the cell suspension was placed on the end of a clean slide, which was previously immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck, Darmstadt, Germany)/ether (Merck) and cleaned with a tissue. The slides were marked with the study identification number and the animal number.
The drop was spread by moving a clean slide with round-whetted sides at an angle of approximately 45° over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol (Merck) and air-dried overnight. At least two slides were prepared per animal.

Staining of the Bone Marrow Smears
The slides were automatically stained using the "Wright-stain-procedure" in a HEMA-tek slide stainer (Hematek 3000, Siemens Healthcare, Den Haag, The Netherlands). This staining is based on Giemsa. The dry slides were automatically embedded in a 1:10 mixture of xylene (Klinipath, Duiven, The Netherlands)/pertex (Klinipath) and mounted with a coverslip in an automated cover slipper (Leica Microsystems B.V., Rijswijk, The Netherlands).

Evaluation criteria:

A micronucleus test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item induces a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes. The positive control data will be analyzed by the Students t test (one-sided, p < 0.05) in case of homogeneous variances or by the Welch t test in case of inhomogeneous variances (one-sided, p < 0.05).
If (one of) the acceptability criteria are not met and the Study Director decides that this has a critical effect on the study, the test will be rejected and repeated.
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Statistics:

ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the data.
A test item is considered positive in the micronucleus test if all of the following criteria are met:
a) At least one of the treatment groups exhibits a statistically significant (one-sided, p <0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test item is considered negative in the micronucleus test if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are within the 95% control limits of the negative historical control data range.
In case the Dunnett’s test shows that there are statistically significant differences between one or more of the test item groups and the vehicle control group a Cochran Armitage trend test (p < 0.05) will be performed to test whether there is a significant trend in the induction.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Dose-range Finding Study
In the dose range finding test, three males and three females were dosed with 2000 mg MLA-3202 (Naugalube OFM 3202)/kg body weight. The animals showed no clinical signs after dosing.

Micronucleus Main Test
Based on the results of the dose-range finding study dose levels of 2000, 1000 and 500 mg/kg body weight were selected as appropriate doses for the micronucleus main test. Since there were no differences between sexes in toxicity only male animals were used in the main study.
Five male animals were used in each treatment group. In addition 15 male mice were treated with the highest dose group and vehicle control group for blood sampling.

Mortality and Toxic Signs
One satellite animal had a bite wound in the genital area before dosing. The animals of the groups treated with MLA-3202 (Naugalube OFM 3202) and the animals of the negative and positive control groups showed no treatment related clinical signs of toxicity or mortality.

Micronucleated Polychromatic Erythrocytes
The mean number of micronucleated polychromatic erythrocytes scored in MLA-3202 (Naugalube OFM 3202) treated groups were compared with the corresponding vehicle control group.
All animals treated with MLA-3202 (Naugalube OFM 3202) exhibited both group mean and individual micronucleated polychromatic erythrocytes which were comparable with both the concurrent vehicle control and the historical vehicle control data. Although animals treated with 1000 mg MLA-3202 (Naugalube OFM 3202)/kg body weight showed a statistically significant increase in the number of micronucleated polychromatic erythrocytes, no statistically significant trend was observed (p=0.111) and the increase was considered not biologically relevant.
The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the within the 95% control limits of the distribution of the historical negative control database.
Cyclophosphamide, the positive control item, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes (Appendix 3). In addition, the number of micronucleated polychromatic erythrocytes found in the positive control animals was within the 95% control limits of the distribution of the historical positive control database.
Hence, all criteria for an acceptable assay were met.

Ratio Polychromatic to Normochromatic Erythrocytes
The groups that were treated with MLA-3202 (Naugalube OFM 3202) showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test item on erythropoiesis.
The group that was treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle control, demonstrating toxic effects on erythropoiesis.

Formulation Analysis
The concentrations analyzed in the formulations of Group B, Group C and Group D were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). No test item was detected in the Group A formulation. The formulations of Group B and Group D were homogeneous (i.e. coefficient of variation ≤ 10%).

Bioanalysis
The objective of this bioanalytical study was to analyze study samples of K2-EDTA mouse plasma for the determination of MLA-3202 (Naugalube OFM 3202) concentrations.
Bioanalysis confirmed the presence of MLA-3202 (Naugalube OFM 3202) in the animals treated with 2000 mg MLA-3202 (Naugalube OFM 3202) per kg body weight in the plasma with a peak 6 hours after treatment..

Mean Body Weight Immediately Prior to First Dosing with MLA-3202 (Naugalube PFM 3202)

Group	Body weight (g) (mean + S.D.)^(1)
A B C D E Satellite A Satellite B	33.6 ± 1.5 34.4 ± 1.8 33.8 ± 1.9 31.2 ± 1.6 34.4 ± 2.1 33.3 ± 2.2 33.9 ± 1.8

(1) Five to ten animals per treatment group

S.D. = standard deviation

 

Mean Body Weight Immediately Prior to Second Dosing with MLA-3202 (Naugalube PFM 3202)

Group	Body weight (g) (mean + S.D.)^(1)
A B C D E Satellite A Satellite B	33.4 ± 0.5 33.8 ± 1.5 34.4 ± 2.3 31.8 ± 1.8 31.8 ± 1.8 33.8 ± 2.6 33.5 ± 1.9

(1) Five to ten animals per treatment group

S.D. = standard deviation

 

Mean Number of Micronucleated Polychromatic Erythrocytes and ratio of Polychromatic/ Normochromatic Erythrocytes

Group	Treatment	Dose (mg/kg body weight)	Number of micronucleated polychromatic erythrocytes (mean ± S.D.)^(1,2)	Ratio polychromatic/ normochromatic erythrocytes (mean ± S.D.)^(1,3)
A B C D E	Vehicle Control Test item Test item Test Item CP	0 2000 1000 500 40	1.0 ± 0.7 1.6 ± 1.5 3.4 ± 2.2^(4) 1.4 ± 1.1 21.0 ± 8.5^(5)	1.04 ± 0.06 0.99 ± 0.04 1.09 ± 0.05 1.07 ± 0.03 0.82 ± 0.05

Vehicle control = Mili-Q water

CP = Cyclophosphamide

(1) Five animals per treatment group

(2) At least 4000 polychromatic erythrocytes were evaluated with a minimum deviation of 5%

(3) The ratio was determined from at least the first 1000 erythrocytes counted

(4) Significantly different from corresponding control group (Dunnett’s t test, P ≤0.05)

(5) Significantly different from corresponding control group (Students t test, Welch t test, P < 0.01)

Conclusions:

In conclusion, MLA-3202 (Naugalube OFM 3202) is not clastogenic or aneugenic in the bone marrow micronucleus test of male mice up to a dose of 2000 mg/kg (the maximum recommended dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.

Executive summary:

The objective of the study was to obtain information on the clastogenicity and aneugenicity of MLA-3202 (Naugalube OFM 3202) when administered to mice at a maximum required acute dose, by measuring the increase in the number of micronucleated polychromatic erythrocytes per 4000 polychromatic erythrocytes in mouse bone marrow.

The study procedures described in this report are in compliance with the most recent OECD and EC guidelines.

Batch RC-1045 of MLA-3202 (Naugalube OFM 3202) was a clear amber-red liquid. The test item was suspended in milli-Q water.

The concentrations analyzed in the formulations of Group B, Group C and Group D were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). No test item was detected in the Group A formulation. The formulations of Group B and Group D were homogeneous (i.e. coefficient of variation ≤ 10%).

In the dose range finding test, three males and three females were dosed with 2000 mg MLA-3202 (Naugalube OFM 3202)/kg body weight. The animals showed no clinical signs after dosing. Since no differences were observed between sexes, the main study was performed with male animals only.

In the main study male animals were dosed twice via oral gavage with vehicle or with 2000, 1000 and 500 mg MLA-3202 (Naugalube OFM 3202) per kg body weight. A positive control group was dosed once via oral gavage with 40 mg cyclophosphamide (CP) per kg body weight. In total 5 treatment groups were used, each consisting of 5 animals. No treatment related clinical signs or mortality were noted in any animal treated with MLA-3202 (Naugalube OFM 3202) or control animals receiving vehicle or cyclophosphamide.

Bone marrow was sampled 48 hours after the first dosing.

No biological relevant increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with MLA-3202 (Naugalube OFM 3202) compared to the vehicle treated animals. All animals treated with MLA-3202 (Naugalube OFM 3202) exhibited both group mean and individual micronucleated polychromatic erythrocytes frequencies which were comparable with both the concurrent vehicle control and the historical vehicle control data.

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the 95% control limits of the distribution of the historical negative control database. Cyclophosphamide, the positive control item, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes.

In addition, the number of micronucleated polychromatic erythrocytes found in the positive control animals was within the 95% control limits of the distribution of the historical positive control database. Hence, all criteria for an acceptable assay were met.

The groups that were treated with MLA-3202 (Naugalube OFM 3202) showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test item on erythropoiesis.

The group that was treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle control, demonstrating toxic effects on erythropoiesis.

Bioanalysis confirmed the presence of MLA-3202 (Naugalube OFM 3202) in the animals treated with 2000 mg MLA-3202 (Naugalube OFM 3202) per kg body weight in the plasma with a peak 6 hours after treatment.

In conclusion, MLA-3202 (Naugalube OFM 3202) is not clastogenic or aneugenic in the bone marrow micronucleus test of male mice up to a dose of 2000 mg/kg (the maximum recommended dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic Toxicity In Vitro

Ames Assay

MLA-3202 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 Aroclor 1254). An additional experiment was performed with tester strain TA98 in the absence of S9-mix, and TA100 in the absence and presence of S9-mix.

 

In the dose range finding test, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. MLA-3202 precipitated on the plates at the dose level of 5000 μg/plate. In tester strain TA100, toxicity was observed in the absence and presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested. Results of this dose range finding test were reported as part of the first mutation assay.

Based on the results of the dose range finding test, the test item was tested in the first mutation assay at a concentration range of 52 to 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test item precipitated on the plates at the top dose of 5000 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.

In a follow-up experiment of the assay with additional parameters, the test item was tested at a concentration range of 492 to 5000 μg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item precipitated on the plates at the top dose of 5000 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in several tester strains, TA1535 (presence of S9-mix), TA1537 (absence of S9-mix), TA98 (absence of S9-mix) and TA100 (absence and presence of S9-mix).

In the second mutation test, not enough non-toxic dose levels were present in the tester strains TA98 (absence of S9-mix) and TA100 (absence and presence of S9-mix), therefore an additional experiment was performed. In this mutation experiment, the test item was tested at a concentration range of 17 to 5000 μg/plate in the absence of S9-mix in both tester strains, and in the presence of 10% (v/v) S9-mix in tester strain TA100. The test item precipitated on the plates at dose level of 5000 μg/plate. In tester strain TA100, toxicity was observed in the absence and presence of S9-mix. In tester strain TA98, no toxicity was observed at any of the dose levels tested.

MLA-3202 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 the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

 

Based on the results of this study it is concluded that MLA-3202 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Chromosome Aberration Assay 

The report describes the effect of MLA-3202 on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix). The possible clastogenicity of MLA-3202 was tested in two independent experiments.

 

In the first cytogenetic assay, MLA-3202 was tested up to 164 μg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction. MLA-3202 precipitated in the culture medium at this dose level.

In the second cytogenetic assay, MLA-3202 was tested up to 70 μg/ml for a 24 h continuous exposure time with a 24 h fixation time and up to 50 μg/ml for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at these dose levels.

 

The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

 

MLA-3202 did not induce any statistically significant and/or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments.

No effects of MLA-3202 on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that MLA-3202 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

 

Finally, it is concluded that this test is valid and that MLA-3202 is not clastogenic in human lymphocytes under the experimental conditions described in the report.

Genetic Toxicity In Vitro

Mammalian Cell Gene Mutation Test

The objective of this study was to evaluate the mutagenic potential of MLA-3202 by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix). The TK mutational system detects base pair mutations, frame shift mutations and small deletions.

 

The test was performed in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period.

Batch RC-1045 of MLA-3202 was a clear amber-red liquid. The test item was dissolved in dimethyl sulfoxide.

 

In the first experiment, MLA-3202 was tested up to concentrations of 35 and 90 μg/ml in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. The relative total growth (RTG) was 1 and 16% in the absence and presence of S9-mix, respectively.

MLA-3202 precipitated in the culture medium at the dose level of 90 μg/ml.

In the second experiment, MLA-3202 was tested up to concentrations of 22.5 μg/ml in the absence of S9-mix. The incubation time was 24 hours. The RTG was 15%. MLA-3202 did not precipitate in the culture medium at this dose level.

 

In the absence of S9-mix, MLA-3202 did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment.

In the presence of S9-mix, MLA-3202 did not induce a significant increase in the mutation frequency.

 

In conclusion, MLA-3202 is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

In Vivo

Micronucleus Assay

The objective of the study was to obtain information on the clastogenicity and aneugenicity of MLA-3202 (Naugalube OFM 3202) when administered to mice at a maximum required acute dose, by measuring the increase in the number of micronucleated polychromatic erythrocytes per 4000 polychromatic erythrocytes in mouse bone marrow.

Since no differences were observed between sexes, the main study was performed with male animals only.

In the main study male animals were dosed twice via oral gavage with vehicle or with 2000, 1000 and 500 mg MLA-3202 (Naugalube OFM 3202) per kg body weight. A positive control group was dosed once via oral gavage with 40 mg cyclophosphamide (CP) per kg body weight. In total 5 treatment groups were used, each consisting of 5 animals. No treatment related clinical signs or mortality were noted in any animal treated with MLA-3202 (Naugalube OFM 3202) or control animals receiving vehicle or cyclophosphamide.

Bone marrow was sampled 48 hours after the first dosing.

No biological relevant increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with MLA-3202 (Naugalube OFM 3202) compared to the vehicle treated animals. All animals treated with MLA-3202 (Naugalube OFM 3202) exhibited both group mean and individual micronucleated polychromatic erythrocytes frequencies which were comparable with both the concurrent vehicle control and the historical vehicle control data.

The groups that were treated with MLA-3202 (Naugalube OFM 3202) showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test item on erythropoiesis.

The group that was treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle control, demonstrating toxic effects on erythropoiesis.

Bioanalysis confirmed the presence of MLA-3202 (Naugalube OFM 3202) in the animals treated with 2000 mg MLA-3202 (Naugalube OFM 3202) per kg body weight in the plasma with a peak 6 hours after treatment.

In conclusion, MLA-3202 (Naugalube OFM 3202) is not clastogenic or aneugenic in the bone marrow micronucleus test of male mice up to a dose of 2000 mg/kg (the maximum recommended dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.

Justification for classification or non-classification

Genetic Toxicity In Vitro

Ames Assay

Based on the results of this study it is concluded that MLA-3202 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay, therefore the substance is not classified under CLP as it does not fulfill the criteria for classification.

Chromosome Aberration Assay

Based on the results of this study it is concluded that MLA-3202 is not clastogenic in a chromosome aberration in cultured human lymphocyte cells study, therefore the substance is not classified under CLP as it does not fulfill the criteria for classification.

Mammalian Cell Gene Mutation Test

Based on the results of this study it is concluded MLA-3202 is not mutagenic in the mouse lymphoma L5178Y test system, therefore the substance is not classified under CLP as it does not fulfill the criteria for classification.