Reaction mass of N1,N1'-(methanediyldibenzene-4,1 -diyl)diaryldiamine and 2-{4-[(4-aminophenyl)amino]benzyl}-N4-(4-{4-[(4-aminophenyl)amino]benzyl}phenyl)aryldiamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The in vitro screening tests (bacterial reverse mutation assay and chromosome aberration test) suggested the substance had the potential for clasotgenicity (OECD 471 and OECD 473). Thus, a decision to evaluate genotoxicity using in vivo methodologies was supported.

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:

20 August 2014 to 19 September 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine and tryptophan

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Rat liver homogenate metabolising system (10 % liver S9 in standard co-factors)

Test concentrations with justification for top dose:

- Experiment 1: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
- Experiment 2: 1.5, 5, 15, 50, 150, 500 and 1500 μg/plate

Vehicle / solvent:

Dimethyl sulphoxide

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulphoxide

Positive controls:

yes

Remarks:

ENNG without S9 mix

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

2 µg/plate for WP2uvrA; 3 µg/plate for TA100; 5 µg/plate for TA1535

Positive controls:

yes

Remarks:

9AA without S9 mix

Positive control substance:

9-aminoacridine

Remarks:

80 µg/plate for TA1537

Positive controls:

yes

Remarks:

4NQO without S9 mix

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

0.2 µg/plate for TA98

Positive controls:

yes

Remarks:

2AA with S9 mix

Positive control substance:

other: 2-aminoanthracene

Remarks:

1 µg/plate for TA100; 2 µg/plate for TA1535 and TA1537; 10 µg/plate for WP2uvrA

Positive controls:

yes

Remarks:

BP with S9 mix

Positive control substance:

benzo(a)pyrene

Remarks:

5 µg/plate for TA98

Details on test system and experimental conditions:

TESTER STRAINS
- The five strains of bacteria used are shown in the table below together with their mutations.
- All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain L T2 through mutations in the histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of the uvrB-bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In the strains TA98 and TAI 00~ the R-factor plasmid pKMIOI enhances chemical and UV-induced mutagenesis via an increase in the error-prone repair pathway. The plasmid also confers ampicillin resistance which acts as a convenient marker (Mortelmans and Zeiger, 2000). In addition to a mutation in the tryptophan operon, the E. coli tester strain contains auvrA-DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel, 1976 and Mortelmans and Riccio, 2000).
- The bacteria used in the test were obtained from the University of California, Berkeley, on culture discs, on 04 August 1995 and from the British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987. All of the strains were stored at approximately -196 °C in a Stateboume liquid nitrogen freezer, model SXR 34.
- In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 1408880 10/18) and incubated at 37 °C for approximately I 0 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

TEST ITEM
- The test item was insoluble in sterile distilled water at 50 mg/mL but fully soluble in dimethyl sulphoxide at the same concentration and acetone at 100 mg/mL in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.
- The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 10 minutes at 40 °C on the day of each experiment. No correction was made for purity. All formulations were used within four hours of preparation and were assumed to be stable for this period.
- Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and was reflected in the GLP compliance statement.
- Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10E-4 microns.

CONTROL ITEMS
- Vehicle, negative (untreated) and positive controls were used in parallel with the test item.
- The vehicle control used was dimethyl sulphoxide.

MICROSOMAL ENZYME FRACTION
- Lot No. PB/PNF S9 27 July 2014 was used in the study.
- The S9 Microsomal fraction was prepared in-house from male rats induced with Phenobarbitone/ß-Naphthoflavone at 80/100 mg/kg/day, orally, for 3 days prior to preparation on day 4.
- The S9 homogenate was produced by homogenizing the liver in a 0.15 M KCl solution (1 g liver to 3 mL KCl) followed by centrifugation at 9000 g.
- The protein content of the resultant supernatant was adjusted to 20 mg/mL.
- Aliquots of the supernatant were frozen and stored at approximately -196 °C.
- Prior to use, each batch of S9 was tested for its capability to activate known mutagens in the Ames test.
- The procedure was designed and conducted to cause the minimum suffering or distress to the animals consistent with the scientific objectives and in accordance with the Harlan Laboratories Ltd, Shardlow, UK policy on animal welfare and the requirements of the United Kingdom's Animals (Scientific Procedure) Act 1986 Amendment Regulations 2012.

S9 MIX AND AGAR
- The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.
- The S9 mix contained S9 (5.0 mL); 1.65 M KCl/0.4 M MgCl2 (1.0 mL); 0.1 M glucose-6-phosphate (2.5 mL); 0.1 M NADP (2.0 mL); 0.2 M sodium phosphate buffer pH 7.4 (25.0 mL); sterile distilled water (14.5 mL).
- A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.
- Top agar was prepared using 0.6% Bacto agar (lot number 3218431 04/18) and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from SGL Ltd (lot numbers 37294 09/14 and 37306 09/14).

DOSE SELECTION FOR EXPERIMENT 1
The maximum concentration was 5000 μg/plate (the maximum recommended dose level).
- Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

EXPERIMENT 1 - WITHOUT METABOLIC ACTIVATION
- The appropriate concentration of test item or solvent (0.1 mL) or appropriate positive control (0.1 mL) was added to 2mL of molten trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel-Bonner agar plate.
- Negative (untreated) controls were also performed on the same day as the mutation test.
- Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

EXPERIMENT 1 - WITH METABOLIC ACTIVATION
- The procedure was the same as described previously except that following addition of the test item formulation and bacterial culture, 0.5 mL of 89-mix was added to the molten trace amino-acid supplemented media instead of phosphate buffer.

DOSE SELECTION FOR EXPERIMENT 2
- The results of Experiment 1 determined the Experiment 2 dose range (1.5, 5, 15, 50, 150, 500 and 1500 μg/plate).
- Up to seven test item dose levels were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the toxic limit of the test item following the change in test methodology.

EXPERIMENT 2 - WITHOUT METABOLIC ACTIVATION
- The procedure was the same as described previously.

EXPERIMENT 2 - WITH METABOLIC ACTIVATION
- The procedure was the same as described previously.

INCUBATION AND SCORING
- All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system.
- The plates were viewed microscopically for evidence of thinning (toxicity).

ACCEPTANCE CRITERIA
- The reverse mutation assay may be considered valid if the following criteria are met:
(i) All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al., (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
(ii) All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls). Acceptable ranges are TA 1535 (7 to 40); TA100 (60 to 200); TA1537 (2 to 30); TA98 (8 to 60); WP2uvrA (10 to 60). Combined historical negative and solvent control ranges for 2012 and 2013 are presented in Appendix 2 (attached).
(iii) All tester strain cultures should be in the range of 0.9 to 9 x 10E-09 bacteria per mL.
(iv) Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation. The historical ranges of the positive control reference items for 2012 and 2013 are presented in Appendix 2 (attached).
(v) There should be a minimum of four non-toxic test item dose levels.
(vi) There should be no evidence of excessive contamination.

Evaluation criteria:

EVALUATION CRITERIA
- There are several criteria for determining a positive result. Any one, or all, of the following can be used to determine the overall result of the study:
(i) A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
(ii) A reproducible increase at one or more concentrations.
(iii) Biological relevance against in-house historical control ranges.
(iv) Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
(v) Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by · an out-of-historical range response (Cariello and Piegorsch, 1996) ).
- A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
- Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

MUTATION TEST

- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile. These data are not given in the report.

- Results for the negative controls (spontaneous mutation rates) are presented in Table 1 (attached) and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

- The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 (attached) and Table 4 and Table 5 for Experiment 2 (attached).

- A history profile of vehicle, untreated and positive control values (reference items) is presented in Appendix 2 (attached).

- The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate and the toxic limit selected as the maximum dose in the second mutation test. The test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains initially from 150 μg/plate (TA1537 dosed in the absence of S9-mix) and from 500 μg/plate to all of the remaining tester strains dosed in both the presence and absence of S9-mix. A dark grey colouration was noted from 150 μg/plate (second experiment) and a test item precipitate (greasy in appearance) was noted by eye at and above 500 μg/plate. Neither of these observations prevented the scoring of revertant colonies.

- There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in the first mutation test. Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 2. A small, statistically significant increase in TA98 revertant colony frequency was observed in the presence of S9-mix

at 150 μg/plate in the first mutation test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 150 μg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.6 times the concurrent vehicle control. Further statistically significant increases were also noted, however these responses were accompanied by weakened bacterial background lawns.

Therefore the responses in this instance would be due to additional amino acid being available to the bacteria allowing the cells to undergo several additional cell divisions and presenting as non-revertant colonies.

- All of the positive control chemicals usedinthe test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Conclusions:

The test item was considered to be non-mutagenic under the conditions of this test.

Executive summary:

GUIDELINE

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

 

METHODS

Salmonella typhimuriumstrains TA1535, TA1537, TA98 and TAlOO andEscherichia colistrainWP2uvrAwere treated with the test item using the Ames plate incorporation method at up toeight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was pre-determined and was 1.5 to 5000 μg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was

amended, following the results of Experiment 1, and was 1.5 to 1500 μg/plate. Up to seven test item dose levels were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the toxic limit of the test item.

 

RESULTS

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate and the toxic limit selected as the maximum dose in the second mutation test. The test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains initially from 150 μg/plate (TA1537 dosed in the absence of S9-mix) and from 500 μg/plate to all of the remaining tester strains dosed in both the presence and absence of S9-mix. A dark grey colouration was noted from 150 μg/plate (second experiment) and a test item precipitate (greasy in appearance) was noted by eye at and above 500 μg/plate. Neither of these observations prevented the scoring of revertant colonies. There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in the first mutation test. Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 2. A small, statistically significant increase in T A98 revertant colony frequency was observed in the presence of S9-mix

at 150 μg/plate in the first mutation test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 150 μg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.6 times the concurrent vehicle control. Further statistically significant increases were also noted, however these responses were accompanied by weakened bacterial background lawns.

Therefore the responses in this instance would be due to additional amino acid being available to the bacteria allowing the cells to undergo several additional cell divisions and presenting as non-revertant colonies.

 

CONCLUSION

The test item was considered to be non-mutagenic under the conditions of this test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 September 2014 to 13 January 2015

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

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: 40 CFR 799.9537 TSCA in vitro mammalian chromosome aberration test

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: chromosome aberration

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes:

Remarks:

human

Details on mammalian cell type (if applicable):

Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented in-house with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS), at approximately 37 °C with 5% C02 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbitone/ß-Naphthoflavone induced S9 microsomal fraction

Test concentrations with justification for top dose:

- Preliminary Toxicity Test: 0, 0.156, 0.31, 0.63, 1.25, 2.5, 5, 10, 20 and 40 µg/mL with and without S9 (2 %)
- Experiment 1: 0.156, 0.31, 0.63, 1.25, 2.5, 5 and 10 µg/mL for 4(20)-hour without S9 and 0.156, 0.31, 0.63, 1.25, 2.5, 5 and 10 µg/mL for 4(20)-hour with S9 (2 %)
- Experiment 2: 0.63, 1.25, 2.5, 5, 7.5, 10, 15 and 20 µg/mL for 24-hour without S9 and 0.63, 1.25, 2.5, 5, 6, 8, 10 and 12 µg/mL for 4(20)-hour with S9 (2 %).

Vehicle / solvent:

Dimethyl sulphoxide (DMSO)

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (Sigma, batch SZBE0220V)

Positive controls:

yes

Remarks:

MMC in the absence of S9

Positive control substance:

mitomycin C

Remarks:

0.4 µg/mL for cultures in Experiment 1 and 0.2 µg/mL for cultures in Experiment 2 (Sigma, batch SLBD1982V dissolved in Minimal Essential Medium)

Positive controls:

yes

Remarks:

CP in the presence of S9

Positive control substance:

cyclophosphamide

Remarks:

5 µg/mL for cultures in Experiments 1 and 2 (Acros, batch SLBK2401V dissolved in DMSO)

Details on test system and experimental conditions:

CELLS
- For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection.
- The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The mean value of the AGT for the pool of regular donors used in the laboratory had been determined to be approximately 16 hours under typical experimental exposure conditions.

TEST ITEM
- The test item was considered to be a UVCB mixture and therefore the maximum recommended dose was initially set at 5000 μg/mL.
- The test item was insoluble in MEM at 50 mg/mL but was soluble in dimethyl sulphoxide (DMSO) at 500 mg/mL in solubility checks performed in-house. The test item was accurately weighed, dissolved in DMSO and serial dilutions prepared.
- There was no significant change in pH when the test item was dosed into media (see table below) and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991 ).
- The test item was formulated within two hours of it being applied to the test system and the test item formulations were assumed to be stable. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation because it is not a requirement of the guidelines. This is an exception with regard to GLP and was reflected in the GLP compliance statement.

MICROSOMAL ENZYME FRACTION
- Lot Nos. PB/PNF S9/27/07/14 and S9/12/10/14 were used in the study.
- The S9 Microsomal fraction was prepared in-house from male rats induced with Phenobarbitone/ß-Naphthoflavone at 80/100 mg/kg/day, orally, for 3 days prior to preparation on day 4.
- The S9 homogenate was produced by homogenizing the liver in a 0.15M KCl solution (1 g liver to 3 mL KCl) followed by centrifugation at 9000 g.
- The protein content of the resultant supernatant was adjusted to 20 mg/mL. Aliquots of the supernatant were frozen and stored at approximately -196 °C.
- Prior to use, each batch of S9 was tested for its capability to activate known mutagens in the Ames test.
- The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20 % (v/v)), MgCl2 (8mM), KCl (33 mM), sodium orthophosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM) and NADP (5 mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2% in the Preliminary Toxicity Test, Experiment 1 and Experiment 2.
- The procedure was designed and conducted to cause the minimum suffering or distress to the animals consistent with the scientific objectives and in accordance with the Envigo Research Limited, Shardlow, UK policy on animal welfare and the requirements of the United Kingdom's Animals (Scientific Procedure) Act 1986 Amendment Regulations 2012.

CULTURE CONDITIONS
- Duplicate lymphocyte cultures (A and B) were established for each dose level.
- Each culture dispensed into plastic flasks contained MEM 10 % FBS (9.05 mL); Li-heparin (0.1 mL); phytohaemagglutinin (0.1 mL); heparinised whole blood (0.75 mL).

WITH METABOLIC ACTIVATION (S9) TREATMENT
- After incubation for approximately 48 hours at approximately 37 °C, 5% C02 in humidified air, the cultures were transferred to tubes and centrifuged.
- Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 0.1 mL of the appropriate solution of vehicle control or test item was added to each culture.
- For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1 mL of 20% S9-mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test, Experiment 1 and Experiment 2.
- The final S9 concentration of 2% was repeated in the second experiment to ascertain if the increase in the frequency of cells with aberrations in the Experiment 1 was reproducible.
- After 4 hours at approximately 37 °C, 5% C02 in humidified air the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium.
- After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at, approximately 37 °C in 5% C02 in humidified air.

WITHOUT METABOLIC ACTIVATION (S9) TREATMENT
- In Experiment 1, after approximately 48 hours incubation at approximately 37 °C with 5% C02 in humidified air the cultures were decanted into tubes and centrifuged.
- Approximately 9 mL of the culture medium was removed and reserved. The cells were then re-suspended in the required volume of fresh MEM (including serum) and dosed with 0.1 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The total volume for each culture was a nominal 10 mL.
- After 4 hours at approximately 37 °C, 5% C02 in humidified air the cultures were centrifuged the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium. The cells were then returned to the incubator for a further 20 hours.
- In Experiment 2, in the absence of metabolic activation, the exposure was continuous for 24 hours. Therefore, when the cultures were established the culture volume was a nominal 9.9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.1 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal final volume of each culture was 10 mL. The cultures were then incubated at approximately 3 7 °C, 5% C02 in humidified air for 24 hours.
- The preliminary toxicity test was performed using both of the exposure conditions as described for Experiment 1 and for Experiment 2 in the absence of metabolic activation only.

PRELIMINARY TOXICITY TEST
- Three exposure groups were used:
(i) 4 hours exposure to the test item without S9-mix, followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
(ii) 4 hours exposure to the test item with S9-mix (2%), followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
(iii) 24-hour continuous exposure to the test item without S9-mix.
- The initial dose range of test item used was 19.53 to 5000 μg/mL. However, with no surviving dose levels due to toxicity it was repeated using a revised dose range of 0.156 to 40 μg/mL.
- Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.
- Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for mitotic index evaluation. Mitotic index data was used to estimate test item toxicity and for selection of the dose levels for the main test.

EXPERIMENT 1
- Two exposure groups were used:
(i) 4-hour exposure to the test item without S9-mix, followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 0.156 to 10 μg/mL.
(ii) 4-hour exposure to the test item with S9-mix (2%), followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 0.156 to 10 μg/mL.

EXPERIMENT 2
- Two exposure groups were used:
(i) 24-hour continuous exposure to the test item without S9-mix prior to cell harvest. The dose range of test item used was 0.63 to 20 μg/mL.
(ii) 4-hour exposure to the test item with S9-mix (2%) followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 0.63 to 12 μg/mL.

CELL HARVEST
- Mitosis was arrested by addition of demecolcine (Colcemid 0.1 μg/mL) two hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl.
- After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCI cell suspension into fresh methanol/glacial acetic acid (3: 1 v/v).
- The fixative was changed at least three times and the cells stored at approximately 4 °C to ensure complete fixation prior to slide preparation.

PREPARATION OF METAPHASE SPREADS
- The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and resuspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labelled with the appropriate identification data.

STAINING
- When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

QUALITATIVE SLIDE ASSESSMENT
- The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for mitotic index evaluation.

CODING
- The slides were coded using a computerized random number generator. Supplementary slides were coded manually.

MITOTIC INDEX
- A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

SCORING OF CHROMOSOME DAMAGE
- Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were at least 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing and the ISCN (1985) (see Appendix 1, attached). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.
- In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported. If a weak or equivocal polyploidy effect was observed then an additional independent evaluation of the frequency of polyploidy cells was considered. In addition, the incidence of cells with endoreduplicated chromosomes was also reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors. The current historical range is shown in Appendix 1 (attached).

CRITERIA FOR DETERMINING THE STUDY CONCLUSION
- A test item can be classified as non-genotoxic if (i) The number of induced chromosome aberrations in all evaluated dose groups is within the range of laboratory historical control data. (ii) No toxicologically or statistically significant increase of the number of structural chromosome aberrations is observed following statistical analysis.
- A test item can be classified as genotoxic if (i) The number of induced structural chromosome aberrations is not in the range of laboratory historical control data and (ii) either a concentration-related or a statistically significant increase of the number of structural chromosome aberrations is observed. Marked increases only observed in one
dose level will be assessed on a case by case basis.
- Biological relevance of the results will be considered first. Statistical methods will be used to analyse the increases in aberration data as recommended in the OECD 4 73 guideline. However, statistical significance will not be the only determining factor for a positive response.
- A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.

Evaluation criteria:

EVALUATION CRITERIA
- Negative control: The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures will normally be within the laboratory historical control data range.
- Positive control: All the positive control chemicals must induce a clear positive response (p ≤ 0.01). Acceptable positive responses demonstrate the validity of the experiment and the integrity of the S9-mix.

Statistics:

STATISTICAL ANALYSIS
- The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. (Richardson et al. 1989).

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

EXPERIMENT 1 - DOSE LEVELS OF CONTROL AND TEST ITEM

Group	Final concentration of test item (µg/mL)
4(20)-hour without S9	0*, 0.156, 0.31, 0.63*, 1.25*, 2.5*, 5*, 10, MMC 0.4*
4(20)-hour with S9 (2 %)	0*, 0.156, 0.31, 0.63, 1.25*, 2.5*, 5*, 10*, CP 5*

* = Dose levels selected for metaphase analysis

MMC = Mitomycin C

CP = Cyclophosphamide

 

EXPERIMENT 2 - DOSE LEVELS OF CONTROL AND TEST ITEM

Group	Final concentration of test item (µg/mL)
24-hour without S9	0*, 0.63, 1.25, 2.5, 5*, 7.5*, 10*, 15*, 20, MMC 0.2*
4(20)-hour with S9 (2 %)	0*, 0.63, 1.25, 2.5, 5*, 6*, 8*, 10, 12, CP 5*

* = Dose levels selected for metaphase analysis

MMC = Mitomycin C

CP = Cyclophosphamide

Conclusions:

The test item induced a statistically significant increase in the frequency of cells with chromosome aberrations, in the absence of a liver enzyme metabolizing system following a 24-hours continuous exposure. The test item was therefore considered to be clastogenic to human lymphocytes in vitro.

Executive summary:

GUIDELINE

Structural chromosomal aberrations were investigated in cultured mammalian cells in accordance with OECD Guidelines for Testing of Chemicals No 473 "In Vitro Mammalian Chromosome Aberration Test", Method B.10 of Commission Regulation (EC) No 440/2008 of 30 May 2008, US EPA OPPTS 870.5375 guideline, 40 CFR 799.9537 TSCA in vitro mammalian chromosome aberration test and guidelines published by the Japanese Ministry of Economy, Trade and Industry (METI), Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries.

 

METHODS

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study; i.e. in Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4 hours exposure with addition of S9 was repeated (using a 2% final S9 concentration) to investigate the reproducibility of the initial response, whilst in the absence of metabolic activation the exposure time was increased to 24 hours. The dose levels used in the main experiments were selected using data from the preliminary toxicity test and were 0.156, 0.31, 0.63, 1.25, 2.5, 5 and 10 µg/mL for 4(20)-hour without S9; 0.156, 0.31, 0.63, 1.25, 2.5, 5 and 10 µg/mL for 4(20)-hour with S9 (2 %); 0.63, 1.25, 2.5, 5, 7.5, 10, 15 and 20 µg/mL for 24-hour without S9; 0.63, 1.25, 2.5, 5, 6, 8, 10 and 12 µg/mL for 4(20)-hour with S9 (2 %).

 

RESULTS

All vehicle (DMSO) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected. The test item was toxic and induced statistically significant increases in the frequency of cells with aberrations, in two of the 4 exposure groups tested, using dose ranges that included a dose levels that induced near optimum mitotic inhibition. In Experiment 1 statistically significant increases in the frequency of aberrations were observed in the 4(20)-hour exposure with metabolic activation; and in Experiment 2 in the 24-hour continuous exposure group.

 

CONCLUSION

The test item was considered to be clastogenic to human lymphocytes in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

26 September 2008 - 16 October 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with minor deviations from standard test guidelines which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

The temperature was recorded to be outside the range of 37.0 ± 1.0 C specified in the protocol for approximately 75 minutes in the second mutation experiment (with a maximum of 38.4 C). The study integrity was not adversely affected by the deviation.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

The Salmonella typhimurium strains used in this study were TA1535, TA1537, TA98 and TA100. The Escherichia coli strain used was WP,uvrA. The strains TA1537 and TA98 are capable of detecting frame shift mutagens, strains TA1535, TA100 and WP,uvrA are capable of detecting base-pair substitution mutagens

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

A liver microsomal preparation (S9-mix) prepared from rats pre-treated with a mixture known to induce an elevated level of these enzymes.

Test concentrations with justification for top dose:

Experiment 1: 3, 10, 33, 100, 333, 1000, 3330 and 5000 ug/plate
Experiment 2: 10, 33, 100, 333, 1000 ug/plate

Vehicle / solvent:

-Vehicle(s)/solvent(s) used: dimethyl sulphoxide
- Justification for choice of solvent/vehicle: no data available.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Preincubation period: not applicable.
- Exposure duration: approximately 48 hours incubation at 37°C
- Expression time (cells in growth medium): at 37°C for approximately 10 hours
- Selection time (if incubation with a selection agent): Not applicable.
- Fixation time (start of exposure up to fixation or harvest of cells): Not applicable.

SELECTION AGENT (mutation assays): Not applicable.
SPINDLE INHIBITOR (cytogenetic assays): Not applicable.
STAIN (for cytogenetic assays): Not applicable.

NUMBER OF REPLICATIONS: Triplicate.

NUMBER OF CELLS EVALUATED: 108.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: the number of revertant colonies

OTHER EXAMINATIONS:
- Determination of polyploidy: not applicable.
- Determination of endoreplication: not applicable.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:

a) The total number of revertants in tester strain TA100 is not greater than two (2) times the
concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WPzuvrA is not greater than three (3) 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 TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP,uvrA is greater than three (3) 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.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: strain/cell type: S. typhimurium TA 1535

Remarks:

Migrated from field 'Test system'.

Relevant results are included in attachments:

Table 1: Experiment I: Mutagenic response of test substance in the Salmonella typhimurium reverse  mutation assay and in the Escherichia coli reverse mutation assay.

Table 2: Experiment 2: Mutagenic response of test substance in the typhimurium reverse mutation assay and in the Escherichia coli.

Table 3: Experiment 3: Mutagenic response of test substance in the typhimurium reverse mutation assay.

Conclusions:

The test material is mutagenic in tester strain TA98 of the Salmonella typhimurium reverse mutation assay. The substance is not mutagenic in the other Salmonella typhimurium tester strains (TA1535, TA1537 or TA100) or Escherichia coli strain reverse mutation assay using strain WP2uvrA. The mutagenicity was confined only to incubations with metabolic activation and at precipitating concentrations.

Executive summary:

Introduction.The method was designed to meet the requirements of the most recent OECD and EEC guidelines.

Methods. The test substance was tested in the Salmonella typhimuriurn 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 R-naphthoflavone). An additional experiment was performed with tester strain TA98 in the presence of S9-mix.

Results. At a concentration of 50 mg/ml, the test material was suspended in dimethyl sulfoxide. At concentrations of 33.3 mg/ml and lower, the test substance was dissolved in dimethyl sulfoxide.

In the dose range finding test, the substance was tested up to concentrations of 5000 ug/plate in the absence and presence of S9-mix in the strains TA100 and WP₂uvrA. The substance precipitated on the plates at dose levels of 1000 ug/plate and upwards. No biologically relevant decrease in the number of revertants was observed at all dose levels tested. No reduction of the bacterial background lawn was observed up to the

dose level of 1000 ug/plate. Due to heavy precipitate the bacterial background lawn could not be determined at concentrations of 3330 and 5000 ug/plate. Results of this dose range finding test were reported as part of the first experiment of the mutation assay.

Based on the results of the dose range finding test, the material was tested in the first mutation assay at a concentration range of 10 to 1000 ug/plate in the absence and presence of 5% (vlv) S9-mix in tester strains TA1535, TA1537 and TA98. In an independent repeat of the assay with

additional parameters, the material was tested at the same concentration range as the first assay in the absence and presence of 10% (vlv) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item precipitated on the plates at the doses of 333 and 1000

ug/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.

To obtain more information about the possible mutagenicity of the material, an additional third  experiment was performed with tester strain TA98 in the presence of 10% (vlv) S9-mix. The test substance was tested at a concentration range of 10 to 1000 ug/plate, precipitation on the plates was observed at the dose levels of 333 and 1000 ug/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 this study, 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. In tester strain TA98, the test substance induced an up to 3.3-fold dose related increase in the number of revertant colonies compared to the solvent control after the modification in the composition of  the S9-mix (10% (vlv) S9-mix) in the second experiment. Verification of this result was performed in an additional third experiment, in which the test substance showed an up to 2.2-fold dose-related increase in the number of revertant colonies compared to the solvent control in the presence of 10% (vlv) S9-mix. Although the increase observed in the third experiment was not three-fold, the increase in the number of revertant colonies was observed in two independent repeat experiments, the observed increases were outside our historical control data range and were dose-related, therefore these increases are considered to be biologically relevant and the test substance is considered to be mutagenic in the presence of S9-mix.

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

Based on the results of this study it is concluded that the test substance is mutagenic in tester strain TA98 of the Salmonella typhimurium reverse mutation assay. The substance is not mutagenic in the other Salmonella typhimurium tester strains (TA1535, TA1537 or TA100) or Escherichia coli strain reverse mutation assay using strain WP,uvrA. The mutagenicity was confined only to incubations with metabolic activation and at precipitating concentrations.

Reference 4

Endpoint:

genetic toxicity in vitro, other

Type of information:

other: expert analysis

Adequacy of study:

supporting study

Study period:

09 November 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline required

Principles of method if other than guideline:

Expert analysis of in vitro genetic toxicity results

GLP compliance:

no

Conclusions:

The in vitro screening tests suggested the substance had the potential for clasotogenicity. Thus, a decision to evaluate genotoxicity using in vivo methodologies was supported.

Executive summary:

Bacterial Reverse Mutation Test – OECD No. 471

Principle

The bacterial reverse mutation test uses amino-acid requiring strains of Salmonella typhimurium and Escherichia coli to detect point mutations, which involve substitution, addition or deletion of one or a few DNA base pairs. The test is not intended to determine conclusively if a substance is genetically toxic to animals or humans but should be considered a screening method used to determine the need for further evaluation.

 

The test detects mutations that restore the functional capability of the bacteria to synthesize an essential amino acid that is not present in the substrate. In other words, the revertant bacteria are detected by their ability to grow in the absence of the amino acid histidine (for S. typhimurium) or tryptophan (for E. coli) required by the parent test strain.

 

Method

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using the Ames plate incorporation method at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors).

 

Two experiments were performed. The dose range for Experiment 1 was 1.5 to 5000 μg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. Toxicity was found to be excessive. The dose range was amended, following the results of Experiment 1, and was 1.5 to 1500 μg/plate. Up to seven test item dose levels were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the toxic limit of the test item.

 

Results

There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in the first mutation test. Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, eitherwith or without metabolic activation in Experiment 2. A small, statistically significant increase in TA98 revertant colony frequency was observed in the presence of S9-mix at 150 μg/plate in the first mutation test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 150 μg/plate were within the in-house historical untreated/vehicle control range for the tester strain and only 1.6 times the concurrent vehicle control.

 

Further statistically significant increases were also noted, however these responses were accompanied by weakened bacterial background lawns. Therefore the responses in this instance would be due to additional amino acid being available to the bacteria allowing the cells to undergo several additional cell divisions and presenting as non-revertant colonies.

 

Expert conclusion

The test item is non-mutagenic under the conditions of thisin vitrotest.

 

In Vitro Mammalian Chromosome Aberration Test–OECD No. 473

Principle

Thein vitrochromosome aberration test identifies substances that cause structural chromosome aberrations in cultured mammalian cells. Structural aberrations may be either chromosomal or chromatid derived. Chromatid-type aberrations occur where breaks and re-joining affects only one of the sister-chromatids at any one locus. The majority of chemical mutagens induce chromatid aberrations type. Alternatively, chromosome-type aberrations occur where breaks and re-joining always affect both sister-chromatids at any one locus. Chromosome mutations are of greater concern since they may cause many genetic diseases in humans and experimental animals. Regardless of the result, it is important to note that the method is not intended to conclusively determine if the substance will cause chromosomal damage. It should be used as a screening method to decide if further genetic testing is required.

 

Method

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study; i.e. in Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4 hour exposure period in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4 hour exposure with addition of S9 was repeated (using a 2% final S9 concentration) to investigate the reproducibility of the initial response, whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

 

Results

The test item was toxic and induced statistically significant increases in the frequency of cells with aberrations, in two of the 4 exposure groups tested. In Experiment 1, statistically significant increases in the frequency of aberrations were observed in the 4 hour exposure (20 hour expression period) with metabolic activation; and in Experiment 2 in the 24-hour continuous exposure group without metabolic activation.

 

Expert conclusion

Based on the results of the in vitro test, that the substance is potentially clastogenic (i.e., an agent that gives rise to or induces disruption or breakages of chromosomes, leading to sections of the chromosome being deleted, added, or rearranged) in the intake animal. However, in evaluating the biological significance of thesein vitroresults, several factors must be considered:

-Screening purpose: This test is meant to be used as ascreen for possible mammalian mutagens and carcinogens. A positive result with thein vitrotest does not categorically indicate that the substance is a mutagen in a whole organism because it does not integrate the benefits of reparative possesses of the intact organism that would typically thwart chromosome damage.

- Potentially poor predictability:in vitrotests that have been followed-up within vivotesting have indicated that substances yielding a positive result in thein vitrostudies are not always indicative ofin vivotesting results [1].

- Cytotoxicity: Although secondary tests at lower doses suggested no indication of cytotoxicity, cytotoxicity leading to positivein vitroresults [2] have resulted in apparent clastogenicity.

- False positives: The specificity ofin vitromammalian cell genotoxicity assays is low. Theyyield a high incidence of positive results that are not observed in animal genotoxicity tests and can be misleading or generate irrelevant positives that should be viewed with scrutiny [3].

-While the in vitro results suggest the substance appears to be clastogenic under the conditions of the test, it must only be considered to have the “potential to be clastogenic” within an intact organism. Follow on testing using in vivo methods were deemed to be therefore necessary to validate thein vitrochromosomal aberration results.

[1]Doktorova TY, Ates G, Vinken M, Vanhaecke T, Rogiers V. Way forward in case of a false positivein vitrogenotoxicity result for a cosmetic substance?. Toxicol in Vitro. 2014 Feb;28(1):54-9.

 

[2] Hilliard CA, Armstrong MJ, Bradt CI, Hill RB, Greenwood SK, Galloway SM. Chromosome aberrationsin vitrorelated to cytotoxicity of nonmutagenic chemicals and metabolic poisons. Environ Mol Mutagen. 1998;31(4):316-26. 

 

[3] Bryce SM, Bemis JC, Mereness JA, Spellman RA, Moss J, Dickinson D, Schuler MJ, Dertinger SD. Interpretingin vitromicronucleus positive results: simple biomarker matrix discriminates clastogens, aneugens, and misleading positive agents. Environ Mol Mutagen. 2014 Aug;55(7):542-55. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

A mouse micronucleus test (OECD 474) and a Comet assay (OECD 489) were performed in vivo and the test material was shown to be non-genotoxic. Furthermore, rats treated with test item for 90 consecutive days via oral gavage did not show neoplasia in various organs (see section 7.5 of this dossier). Therefore, the test material is unlikely to be genotoxic; no further genotoxicity testing is necessary or recommended.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

single cell gel electrophoresis (comet) assay

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 April 2015 to 05 June 2015

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Deviations:

yes

Remarks:

unathorised necropsies performed during range-finding study with no impact on results or integrity of study (see below)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

ANIMALS AND ANIMAL HUSBANDRY
- Sufficient male Wistar Han (HsdRCCHan WIST) rats were supplied by Envigo RMS (UK) Limited.
- At the start of the main test the males weighed 184.2 to 216.9 g, and were approximately eight to ten weeks old.
- Details of the individual animal weights, group means and standard deviations are presented in Table 1 (attached).
- After a minimum acclimatisation period of five days the animals were selected at random and given a number unique within the study by tail marking and a number written on a colour coded cage card.
- The animals were housed in groups of up to four in solid-floor polypropylene cages with wood-flake bedding.
- Free access to mains drinking water and food (Envigo Teklad 2014C Global Certified Rodent Diet supplied by Envigo RMS Limited, Oxon, UK) was allowed throughout the study. Representative analyses of food and water quality are retained in the laboratory archive.
- The temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70 %, respectively.
- The rate of air exchange was approximately fifteen changes per hour.
- Lighting was controlled by a time switch to give twelve hours light and twelve hours darkness.

Route of administration:

oral: gavage

Vehicle:

VEHICLE CONTROL
- Identification: Arachis oil
- Serial number: V-5957
- Expiry date: 13 June 2016
- Storage conditions: Room temperature

Details on exposure:

TEST ITEM PREPARATION
- The test item was freshly prepared as required as a solution at the appropriate concentration in arachis oil.
- No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulations.
- The test item was formulated within two hours of it being applied to the test system and it was assumed that the formulations were stable for this duration. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

PREPARATION OF POSITIVE CONTROL
- the positive control item, N-Nitroso-N-methylurea, was freshly prepared as required as a solution at the appropriate concentration in distilled water (Laboratoire Aguettant 3011010).

RANGE-FINDING TOXICITY TEST
- A range-finding test was performed to find suitable dose levels of the test item following a double oral administration at zero and 20 hours.
- The upper dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of2000 mg/kg.
- Only male rats were used for the study since there was no marked difference in toxicity demonstrated between the sexes in a previous mouse micronucleus study.
- Groups of male rats were dosed as shown in the table below.
- All animals were dosed twice 20 hours apart at the appropriate dose level by gavage using a metal cannula attached to a graduated syringe.
- The volume administered to each animal was calculated according to its bodyweight at the time of the initial dosing.
- Animals were observed 1 hour after each dosing and immediately prior to termination.
- Any deaths and evidence of overt toxicity were recorded at each observation.

COMET TEST
- Experimental design is summarised in the table below.
- The groups of rats from each dose level were killed 24 hours following the initial treatment.
- Groups each of seven male rats were dosed twice with a 20 hour interval via the oral route with the test item at 1200, 600 or 300 mg/kg.
- The groups of rats from each dose level were killed by humane euthanasia (carbon dioxide asphyxiation) approximately 4 hours following the second administration.
- In addition, two further groups of rats were included in the study; one group (seven male rats) was dosed twice with a 20-hour interval via the oral route with the vehicle alone (arachis oil) and a second group (five male rats) was dosed twice orally with 20-hour interval with N-Nitroso-N-methylurea (MNU) to act as the positive control. MNU is a positive control material that has been shown in-house to produce strand breaks and damage to DNA under the conditions of the test.
- The vehicle and positive control groups of rats were killed by humane euthanasia (carbon dioxide asphyxiation) 24 hours after the start of the test.
- All animals were observed for signs of overt toxicity and death one hour after dosing and then once daily as applicable and immediately prior to termination.

Duration of treatment / exposure:

24 hours

Frequency of treatment:

Two doses (20 hours apart)

Post exposure period:

Not applicable

Dose / conc.:

1 200 mg/kg bw/day (nominal)

Dose / conc.:

600 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Seven

Control animals:

yes, concurrent vehicle

Positive control(s):

POSITIVE CONTROL MATERIAL
- Identification: N-Nitroso-N-methylurea
- Lot number: P102-01944
- Serial number: R-6206
- Purity: 90 %
- Expiry date: 12 April 2017
- Storage conditions: Approximately 4 °C in the dark

Tissues and cell types examined:

- The primary target tissues investigated in this study were liver and glandular stomach.

Details of tissue and slide preparation:

TISSUE SAMPLE REQUIREMENTS
- Humane euthanasia was performed on the animals at the end of the exposure period, using a method that did not affect the integrity of the required tissues. Samples of liver and glandular stomach were obtained from each animal.
- Sub-samples of the primary tissues taken from the vehicle control animals and the dose group animals were processed and preserved for possible histopathology investigations. Assessment of cytotoxicity by histopathology is conducted if the results from the Comet assay, or other observations, suggest cytotoxicity may be confounding the interpretation of the Comet assay.
- The remaining tissue samples were processed to provide single cell suspensions, providing sufficient cells for scoring, for the Comet Assay.
- Liver: A small piece of liver was excised (approximately 1 cm3) and washed in liver buffer, (Hanks balanced salt solution supplemented with EDTA), before being minced and filtered to provide a single cell suspension.
- Glandular Stomach: The stomach was removed and cut longitudinally to allow the stomach contents to be removed. Half the stomach was removed for possible histopathology and the remaining stomach was immersed in stomach buffer (Hanks balanced salt solution supplemented with EDTA and EGTA) and incubated for approximately 15 minutes on ice. The mucosal layer of the stomach was removed by scraping and a single cell suspension was obtained by scraping the underlying glandular stomach tissue and suspending it in stomach buffer. The resulting cell suspension was filtered through gauze prior to use for the comet slides.
- The above procedures were performed under subdued lighting and the Comet Assay tissues/cells were processed at approximately 4 °C.

SLIDE PREPARATION
- Adequate numbers of slides were pre-coated with 0.5 % normal melting point agarose and stored at room temperature prior to the start of the experiment. The slides were labelled for animal number, study number and tissue type prior to use for the comet assay.
- Once the cell suspensions had been obtained, approximately 30 μL was added to 270 μL of 0.5 % low melting point (LMP) agarose, mixed thoroughly and 50 μL of this agarose/cell suspension mix was placed onto a pre-coated slide. Two gels were placed on each slide, and 4 gels were prepared for each tissue. Two of the gels were scored for Comets and two were kept in reserve in case further scoring was required or the gels were damaged during processing. The agarose/cell suspension was immediately covered with a glass coverslip and kept at approximately 4 °C in the dark for approximately 20 minutes to allow it to solidify. All of the slides went through the subsequent processing.
- After the LMP agarose had set the coverslips were removed and the slides gently lowered into freshly prepared lysing solution (pH 10) and refrigerated in the dark for at least 1 hour.
- After the lysis phase had been completed the slides were removed from the lysing solution, briefly rinsed with neutralisation buffer and placed onto the platform of a electrophoresis bath, which was filled with chilled electrophoresis buffer, until the slide surface was just covered. The slides were then left for approximately 20 minutes to allow the DNA to unwind.
- When the DNA unwinding period had finished the slides were subjected to electrophoresis at approximately 0.7 V/cm (calculated between the electrodes), 300 mA for approximately 20 minutes. The buffer in the bath was chilled during the electrophoresis period and the temperature of the electrophoresis buffer was monitored at the start of unwinding, the start of electrophoresis and the end of electrophoresis. The aim was to induce sufficient migration of the DNA so that minimal sized Comets are produced in the nuclei of vehicle control cells.
- At the end of the electrophoresis period the bath was switched off, the slides gently removed and placed on to a draining surface and drop wise coated with a neutralisation buffer and allowed to rest for at least 5 minutes. The slides were then drained and a repeat of the addition of the neutralisation buffer performed twice. The slides were then carefully drained and fixed in cold 100% methanol for 5 minutes and allowed to air dry. Once dry the slides were stored prior to scoring. Two of the four processed slide gels were scored and the remaining slides were stored as backup slides.

HISTOPATHOLOGY
- Samples of the above primary tissues from all animals were preserved in buffered 10% formalin.

TREATMENT OF RESULTS
- Individual animal data and grouped data is presented. The median % DNA for each slide was determined and the mean of the median value calculated for each animal. The mean of the individual animal means is calculated to give a group mean. Alternative methods for calculating and presenting the data may be utilised if considered appropriate.
- When there is no indication of any increase in % Tail Intensity at any dose level then statistical analysis may not be necessary. In all other circumstances comparisons will be made between the appropriate vehicle control value and each individual dose level, using an appropriate statistical method.

ACCEPTABILITY CRITERIA
- The following criteria are used to determine a valid assay:
(i) The concurrent negative control is comparable with the laboratory historical negative control range.
(ii) The positive controls induce responses that are comparable with those in the laboratory positive control range.
(iii) Adequate numbers of cells and doses have been analysed.
(iv) The highest dose level selected meets the requirements of the guideline and the study plan.

Evaluation criteria:

EVALUATION AND INTERPRETION OF RESULTS
- Providing that all the acceptability criteria are fulfilled, a test item is considered to be clearly negative if:
(i) None of the test concentrations exhibits a significant increase compared with the concurrent negative control.
(ii) There is no evidence of a dose-related response.
(iii) The results are within the laboratory historical vehicle control range.
(iv) There is evidence, direct or indirect, to demonstrate exposure or toxicity to the target tissue has been achieved.
- The test item is then considered unable to induce DNA strand breakage in the tissues studied in the test system.
- Providing that all the acceptability criteria are fulfilled, a test item is considered to be clearly positive if:
(i) At least one of the test doses exhibits a statistically significant increase compared to the concurrent negative control.
(ii) The response is considered to be dose related.
(iii) The results are substantially outside the laboratory historical vehicle control range.
- The test item can be considered to induce DNA strand breakage in a particular tissue if all three conditions are met.
- There is no requirement for verification of a clearly positive or negative response.
- Although most experiments will be expected to give clear negative or positive results in rare cases the data set will preclude making a definite judgement. This may require the scoring of additional slides to increase the number of cells and, therefore, add more power to the data. If this does not resolve the issue then the result will be given as equivocal or questionable, and may require the histopathological assessment of the tissues to see if cell toxicity may be the causative agent rather than any genotoxic mechanism.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING TOXICITY TEST
- Mortality data are summarised in the table below.
- In animals dosed with test item there was a premature death at 2000 mg/kg after the second dosing. Clinical signs were also observed at termination at 1200, 1500 and 2000mg/kg and these included hunched posture, lethargy, splayed gait, pilo-erection, diarrhoea, staining around the mouth and decreased respiratory rate. There were no clinical signs seen in the animals dosed with the test item at 1000 mg/kg.
- Based on the above data the maximum tolerated dose (MTD) of the test item, 1200 mg/kg, was selected for use in the main test, with 600 and 300 mg/kg as the lower dose levels. The clinical observations noted at termination of the animals (hunched posture and lethargy) were considered to be acceptable.

COMET ASSAY – MORTALITY DATA AND CLINICAL OBSERVATIONS
- There were no premature deaths seen in any of the test item dose groups.
- Clinical signs were observed in animals dosed with the test item at and above 600 mg/kg and these included
hunched posture and pilo-erection.

EVALUATION OF COMET ASSAY SLIDES
- A summary of the combined group data for each tissue scored for the Cornet Assay (glandular
stomach and liver) is presented in the table below.
- Individual animal data for each tissue are presented in Tables 2 to 11 (attached) with the means, medians and standard deviations calculated from the individual animal data and the mean of median percentage tail intensity group data.
- Historical control data for % tail intensity are presented in Appendix 1 (attached).
- The vehicle control group induced percentage tail intensities which were consistent with the current laboratory historical control range. The positive control material (MNU) produced a marked increase in the percentage tail intensity and median % tail intensity in the liver and glandular stomach. The test method itself was therefore operating as expected and was considered to be valid under the conditions of the test.
- There was no marked increase in percentage tail intensity for any of the test item dose levels in the glandular stomach or the liver tissues when compared to the vehicle control, confirming the test item did not induce DNA damage in the glandular stomach or the liver under the conditions of the test.
- There was no marked increase in hedgehog frequency for any of the test item dose levels in any of the tissues investigated. The hedgehog frequency data for each tissue is included in Tables 2 to 11 (attached).

MORTALITY IN THE RANGE-FINDING TOXICITY TEST

Dose level (mg/kg)	Sex	Number of animals treated	Route	Deaths on day 0	Deaths on day 1
1000	Male	2	Oral	0	0
2000	Male	2	Oral	0	1
1500	Male	2	Oral	0	0
1200	Male	2	Oral	0	0
1200	Male	2	Oral	0	0

COMET TEST –-GLANDULAR STOMACH RESULTS

Dose group	Group mean % hedgehogs	Group mean % tail intensity	Group mean of mean of median % tail intensity per animal
Vehicle	2.75	2.67	1.12
300 mg/kg	4.21	2.57	1.06
600 mg/kg	3.28	1.44	0.36
1200 mg/kg	3.34	1.51	0.33
Positive control (MNU)	6.74	31.37	30.11

 

COMET TEST – LIVER RESULTS

Dose group	Group mean % hedgehogs	Group mean % tail intensity	Group mean of mean of median % tail intensity per animal
Vehicle	0.14	0.52	0.02
300 mg/kg	0.21	0.68	0.03
600 mg/kg	0.00	0.44	0.01
1200 mg/kg	0.21	0.45	0.03
Positive control (MNU)	1.16	21.38	21.28

Conclusions:

The test item did not induce any increases in the % tail intensity values in either the glandular stomach or liver tissue investigated. The test item was considered to be non-genotoxic to the rat tissue investigated in vivo.

Executive summary:

GUIDELINE

The investigation was performed in accordance with OECD Guideline for the Testing of Chemicals No 489 “In vivo Mammalian Alkaline Comet Assay”, adopted 26 September 2014. The Comet Assay has been designed using the recommendations of the International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington DC 1999, as described by Ticeetal.,2000. The primary target tissues investigated in this study were liver and glandular stomach.

 

METHODS

A range-finding test was performed to find suitable dose levels of the test item. The study was performed using only male rats, based on the toxicity response seen in the Mouse Micronucleus Test. Groups, each of seven rats, were dosed twice at 0 and 20 hours with the test item via the oral route. The Comet assay main test was conducted at the maximum tolerated dose (MTD) 1200 mg/kg with 600 mg/kg and 300 mg/kg as the lower dose levels. Animals were killed 4 hours after the second dose administration, the glandular stomach and liver tissues processed and the slides were then prepared and processed prior to scoring for the presence of Comets. Further groups of rats were given a double oral dose of arachis oil (seven rats) or N-Nitroso-N-methylurea (five rats), to serve as vehicle and positive controls respectively.

 

RESULTS

The presence of clinical signs indicated that systemic absorption had occurred. There was no evidence of an increase in the % tail intensity values in animals dosed with the test item dose groups when compared to the concurrent vehicle control group. The positive control material produced a marked increase in the % tail intensity value in all tissues scored, indicating that the test method was working as expected.

 

CONCLUSION

The test item did not induce any increases in the % tail intensity values in either the glandular stomach or liver tissue investigated. The test item was considered to be non-genotoxic to the rat tissue investigated in vivo.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 September 2014 to 16 December 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: damage to chromosomes and/or aneuploidy

Species:

mouse

Strain:

CD-1

Remarks:

albino Hsd: ICR (CD-1)

Sex:

male

Details on test animals or test system and environmental conditions:

ANIMALS AND ANIMAL HUSBANDRY
- Sufficient albino Hsd: ICR (CD-1) strain mice were obtained from Harlan Laboratories UK Ltd, Oxon, UK.
- At the start of the main test, the mice weighed 22.5 to 30.4 g and were approximately six to ten weeks old.
- After a minimum acclimatisation period of five days, the animals were selected at random and given a number unique within the study by tail marking and a number written on a colour coded cage card.
- The animals were housed in groups of up to seven in solid-floor polypropylene cages with wood-flake bedding.
- Free access to mains drinking water and food (Harlan Teklad 2014C Global Certified Rodent diet supplied by Harlan Laboratories Ltd, Oxon, UK) was allowed throughout the study. Representative analyses of food and water quality are retained in the laboratory archive.
- Temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70 % respectively.
- The rate of air exchange was approximately 15 changes per hour.
- Lighting was controlled by a time switch to give twelve hours light and twelve hours darkness.

Route of administration:

intraperitoneal

Vehicle:

- Identification: Arachis oil
- Harlan serial number: V-5957
- Expiry date: 13 June 2016
- Storage conditions: Room temperature

Details on exposure:

PREPARATION OF TEST ITEM
- When required, the test item was freshly prepared as a suspension at the appropriate concentration in arachis oil.
- No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours of it being applied to the test system and it was assumed the formulation was stable over that duration. This is an exception with regard to GLP and was reflected in the GLP compliance statement.

PREPARATION OF POSITIVE CONTROL ITEM
- When required, the positive control item was freshly prepared as a solution at the appropriate concentration in distilled water (Laboratoire Aguettant batch number 3010598).

RANGE-FINDING TOXICITY TEST
- A range-finding toxicity test was performed to determine a suitable dose level and route of administration for the main test.
- The dose level selected should ideally be the maximum tolerated dose level, or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg.
- The range-finding test was also used to determine if the main test should be performed using both sexes or males only.
- Where considered appropriate, bone marrow samples were taken from the range-finding animals. Slides were then prepared and qualitatively assessed to ensure that any bone marrow toxicity observed was within acceptable limits for the main test.
- Groups of mice were dosed as shown in the table below.
- All animals were dosed once only at the appropriate dose level.
- Dosing was by gavage using a metal cannula or with a hypodermic needle attached to a graduated syringe.
- The volume administered to each animal was calculated according to its bodyweight at the time of dosing.
- Animals were observed one hour after dosing and subsequently once daily for two days.
- Any deaths and evidence of overt toxicity were recorded at each observation.
- No necropsies were performed.

MICRONUCLEUS TEST
- Groups of seven mice were dosed with the test item once only via the intraperitoneal route. Doses received were 200, 100 or 50 mg/kg.
- One group of mice from each dose level was killed by cervical dislocation 24 hours following treatment and a second group dosed with test item at 200 mg/kg was killed after 48 hours.
- In addition, two further groups of mice were included in the study; one group (seven mice) was dosed via the oral route with the vehicle alone (arachis oil) and second group (five mice) was dosed orally with cyclophosphamide, which is a positive control item known to produce micronuclei under the conditions of the test.
- The vehicle controls and positive control group animals were killed 24 hours after dosing.
- Experimental design is summarised in the table below.
- All animals were observed one hour after dosing and then once daily and immediately prior to termination.

Duration of treatment / exposure:

24 or 48 hours

Frequency of treatment:

Single dose

Post exposure period:

Not applicable

Dose / conc.:

200 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Seven

Control animals:

yes, concurrent vehicle

Positive control(s):

POSITIVE CONTROL ITEM
- Identification: Cyclophosphamide
- Supplier: Sigma-Aldrich
- Lot number: SLBG4216V
- Harlan serial number: R-6096
- Purity: 97 %
- Expiry date: 13 October 2015
- Storage conditions: Approximately 4 °C in the dark

Tissues and cell types examined:

Both femurs were dissected from each animal immediately following sacrifice.

Details of tissue and slide preparation:

SLIDE PREPARATION
- Immediately following termination (24 or 48 hours after dosing), both femurs were dissected from each animal, aspirated with foetal bovine serum and bone marrow smears prepared following centrifugation and re-suspension.
- The smears were air-dried, fixed in absolute methanol, stained in May-Grünwald/Giemsa, allowed to air-dry and a cover slip was applied using mounting medium.

Evaluation criteria:

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

INTERPRETATION OF RESULTS
- A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test item groups and the number occurring in the vehicle control group.
- A positive mutagenic response was demonstrated when a statistically significant, dose-responsive, toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48-hour kill times when compared to the vehicle control group.
- If these criteria were not fulfilled, then the test item was considered to be non-genotoxic under the conditions of the test.
- A positive response for bone marrow toxicity was demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the vehicle control group.

Statistics:

STATISTICAL ANALYSIS
- All data were statistically analysed using appropriate methods recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989).
- Data were analysed following a square root of (x + 1) transformation using Student’s t-test (two tailed) and any significant results were confirmed using the one way analysis of variance.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING TOXICITY TEST
- Mortality data are summarised in the table below.
- Clinical signs were not observed in any of the animals dosed with the test item at 1000 or 2000 mg/kg via the oral route. Qualitative assessment of the slides prepared from 2000 mg/kg range-finding animals revealed no evidence of any toxicity to the bone marrow. Therefore, with no evidence of exposure to the target tissue, and following consultation with the sponsor, the intraperitoneal route was investigated to maximise the potential exposure of the test item to the animals.
- Clinical signs observed in one of the animals dosed with the test item at 1000 mg/kg via the intraperitoneal route were hunched posture, ptosis, splayed gait and lethargy. A premature death was also observed in one animal. It should also be noted that the premature death and clinical signs were only observed at the 48 hour post-dosing observations.
- In animals dosed with the test item at 600 mg/kg via the intraperitoneal route, clinical signs of hunched posture and ptosis were seen and a premature death was observed at the 48-hour post-dosing observations.
- In animals dosed with the test item at 400 mg/kg via the intraperitoneal route, clinical signs of hunched posture, ptosis and emaciation were observed.
- Clinical signs were not observed in either of the animals dosed with the test item at 200 mg/kg.
- In one of the animals dosed with test item at 300 mg/kg, clinical signs of hunched posture, ptosis and emaciation were observed at the 48 hour observations.
- Qualitative assessment of the slides prepared from the 300 mg/kg range-finding animals also revealed evidence of toxicity to the bone marrow. Therefore with evidence of excessive toxicity at and above 300 mg/kg, confirmatory animals were dosed with the test item at 200 mg/kg and clinical signs were once again not observed. Qualitative analysis of the slides prepared from the confirmatory animals indicated modest but acceptable levels of toxicity to the bone marrow.
- The test item showed no marked differences in its toxicity to male or female mice and it was considered acceptable to use males only for the main test. The maximum tolerated dose (MTD) of the test item was considered to be 200 mg/kg and this was selected for use in the main test. Dose levels of 100 and 50 mg/kg were selected as the lower dose levels and the intraperitoneal route of administration was used for all three dose groups.

MICRONUCLEUS TEST
- There were no premature deaths or clinical signs observed in any of the dose groups in the main test.

EVALUATION OF BONE MARROW SLIDES
- A summary of results of the micronucleus test is given in Table 1 (attached).
- Individual and group mean data are presented in Tables 2 to 7 (attached).
- Historical control data from 2011, 2012 and 2013 are presented in Appendix 1 (attached).
- No statistically significant decreases in the PCE/NCE ratio were observed in the 24 or 48-hour test material dose groups when compared to the vehicle control group.
- There were no statistically significant increases in the frequency of micronucleated PCEs in any of the test material dose groups when compared to the vehicle control group.
- The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

MORTALITY DURING RANGE-FINDING TOXICITY TEST

Dose level (mg/kg)	Sex	Number of animals treated	Route	Deaths on day 0	Deaths on day 1	Deaths on day 2
1000	Male	1	Oral	0	0	0
1000	Female	1	Oral	0	0	0
2000	Male	1	Oral	0	0	0
2000	Female	1	Oral	0	0	0
1000	Male	1	ip	0	0	1
1000	Female	1	ip	0	0	0
600	Male	1	ip	0	0	0
600	Female	1	ip	0	0	1
400	Male	1	ip	0	0	0
400	Female	1	ip	0	0	0
200	Male	1	ip	0	0	0
200	Female	1	ip	0	0	0
300	Male	1	ip	0	0	0
300	Female	1	ip	0	0	0
200	Male	2	Ip	0	0	0

Conclusions:

The test item was considered to be non-genotoxic under the conditions of the test.

Executive summary:

GUIDELINE

The study was performed to assess the potential of the test item to produce damage to chromosomes or aneuploidy when administered to mice. The method was designed to be compatible with the 1997 OECD Guidelines for Testing of Chemicals No 474 “Mammalian Erythrocyte Micronucleus Test”, Method B.12 of Commission Regulation (EC) No 440/2008 of 30 May 2008, the US EPA (TSCA) OPPTS 870.5395, EPA 712-C-98-226, August 1998 guidelines, and be acceptable to the Japanese METI/MHLW/MAFF guidelines for testing of new chemical susbtances.

 

METHODS

A range-finding test was performed to identify suitable doses levels of the test item and route of administration and to investigate if there was a marked difference in toxic response between the sexes. There was no marked difference in test item toxicity between the sexes and the main test was performed using only male mice. The micronucleus test was conducted using the intraperitoneal route in groups of seven mice (males) at the maximum tolerated dose of 200 mg/kg with 100 and 50 mg/kg as the two lower dose levels. Animals were killed 24 or 48 hours later, the bone marrow extracted, and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei. Additional groups of mice were given a single oral dose of arachis oil (7 mice) or dosed orally with cyclophosphamide (5 mice) to serve as vehicle and positive controls respectively. Vehicle and positive control animals were killed 24 hours later.

 

RESULTS

There were no premature deaths or clinical signs observed in any of the dose groups in the main test. No statistically significant decreases in the PCE/NCE ratio were observed in the 24 or 48-hour test material dose groups when compared to the vehicle control group. There were no statistically significant increases in the frequency of micronucleated PCEs in any of the test material dose groups compared to the vehicle control group. The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

 

CONCLUSION

The test item was considered to be non-genotoxic under the conditions of the test.

Reference 3

Endpoint:

genetic toxicity in vivo, other

Type of information:

other: expert analysis

Adequacy of study:

supporting study

Study period:

09 November 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline required

Principles of method if other than guideline:

Expert analysis of in vivo genetic toxicity results

GLP compliance:

no

Conclusions:

Two in vivo studies were conducted and the test material was not found to be genotoxic. Furthermore, rats treated with test item for 90 consecutive days via oral gavage did not show neoplasia in various organs (see section 7.5 of this dossier). Therefore, the test material is unlikely to be genotoxic; no further genotoxicity testing is necessary or recommended.

Executive summary:

In vivo Mammalian Erythrocyte Micronucleus Test – OECD No. 474

Principle

The purpose of the micronucleus test is to identify substances that cause cytogenetic damage in live animals which results in the formation of micronuclei containing either lagging chromosome fragments or whole chromosomes by damaging the mitotic apparatus of erythroblasts. The test is especially relevant for assessing genotoxicity becausein vivometabolism, pharmacokinetics and DNA repair processes are active in the whole animal and contribute to the responses.

 

Method

A range-finding test determined suitable dose levels of the test item and the potential for marked differences in the toxic response between the sexes. There was no marked gender difference in toxicity of the test item so the main test was performed using male mice. Animals were dosed via the intraperitoneal route in groups of seven at the 200 mg/kg (the maximum tolerated dose), 100 and 50 mg/kg and euthanized at 24 or 48 hours. The bone marrow was extracted, and smears were prepared and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei indicative of chromosomal damage. Negative controls (7 mice) were given a single oral dose of arachis oil. Positive controls (5 mice) were dosed orally with cyclophosphamide.

 

Results

In the intact organism, there were no statistically significant increases in the frequency of micronucleated PCEs in any of the test material dose groups when compared to the vehicle control group. The positive control group showed an increase in the incidence of micronucleated polychromatic erythrocytes confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide. No statistically significant decreases in the PCE/NCE ratio were observed in the 24 or 48-hour dosing groups when compared to the vehicle control group.

 

Expert conclusion

The results of this in vivo test demonstrate that the substance is not cytogenic in the whole organism. This is especially significant given thein vitroscreening chromosomal aberration test results that suggested otherwise. Therefore, because thein vivotesting incorporates animal pharmacokinetics/pharmacodynamics and DNA repair processes that are active in the whole animal, it must be given greater emphasis in a weight of evidence evaluation of intrinsic and biologically relevant substance cytogenicity.

 

In vivoMammalian Alkaline Comet Assay – OECD No. 489

Principle

Intrinsic mutagenic hazard of a substance must demonstrate DNA damage in intact animals. As noted above, thein vivomicronucleus test results indicate no damage to bone marrow cells of mice and add substantially to the weight of evidence that the substance is not inherently clastogenic. Using the Comet Assay, additionalin vivotesting was performed to verify the micronucleus results.

 

Thein vivoalkaline comet (single cell gel electrophoresis) assay detects DNA strand breaks in cells or nuclei isolated from multiple tissues of rats that have been exposed to the test substance. The test is designed to ensure that the animals have been exposed to the substance over a sufficient period of time such that it is distributed throughout the organism and is subject to metabolic processes that may lead to DNA damage as a fixed stable mutation or to repair resulting in no persistent effect.

 

Method

Male rats were used for the study since there was no marked difference in toxicity between the sexes in the above discussed previous mouse micronucleus study. Rats were administered arachis oil as a negative control and N-Nitroso-N-methylurea, in water, as a positive control. Rats subject to substance treatment were dosed twice within a 20 hour interval via gavage at 300, 600, and 1200 mg/kg in arachis oil. Rats from each dose level and controls were euthanized 24 hours following the initial treatment.

 

Samples of liver and glandular stomach were obtained from each animal. Cell suspensions were filtered through gauze prior to use for the comet slides. Once the cell suspensions had been obtained, approximately 30 μL was added to 270 μL of 0.5% low melting point (LMP) agarose, mixed thoroughly and 50 μL of this agarose/cell suspension mix was placed onto pre-coated slides. Two gels were placed on each slide, and 4 gels were prepared for each tissue (liver and glandular stomach).

 

The slides were placed into a lysing solution (pH 10) and refrigerated in the dark for at least 1 hour after which they were removed, rinsed with neutralization buffer, and placed into an electrophoresis bath for approximately 20 minutes to allow the DNA to unwind. DNA was subjected to electrophoresis at approximately 0.7 V/cm, 300 mA for approximately 20 minutes. The slides were removed and suspensions were neutralized, fixed in cold 100% methanol for 5 minutes and allowed to air dry.

 

Comet slides were hydrated, stained and placed onto the stage of a fluorescence microscope and scored for fluorescent comets. The primary endpoint was percentage DNA in the tail (percentage tail intensity). However, each slide was also assessed for the incidence of heavily damaged cells otherwise referred to as ‘hedgehog’ cells.

 

The median % DNA for each slide was determined the mean of the individual animal means was calculated to give a group mean. A statistical comparison was made between the appropriate vehicle control value and each individual dose level to determine if DNA damage was observed to be associated with exposure to the substance.

 

Results

There were no statistically significant increases in the group mean % tail intensity. This shows that the substancein vivodid not induce DNA damage in the cells of the glandular stomach (target tissue at the site of administration) or in the cells of the liver (target tissue at the primary site of metabolism after absorption).

 

Expert conclusion

The in vivo Comet assay did not indicate any DNA damage to the living organism. Therefore it should be considered to be non-genotoxic. These results corroborate those of the in vivo MNA and provide further evidence that the substance is not inherently genotoxic to the intact organism.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Bacterial Reverse Mutation Test – OECD No. 471

Principle

The bacterial reverse mutation test uses amino-acid requiring strains of Salmonella typhimurium and Escherichia coli to detect point mutations, which involve substitution, addition or deletion of one or a few DNA base pairs. The test is not intended to determine conclusively if a substance is genetically toxic to animals or humans but should be considered a screening method used to determine the need for further evaluation. The test detects mutations that restore the functional capability of the bacteria to synthesize an essential amino acid that is not present in the substrate. In other words, the revertant bacteria are detected by their ability to grow in the absence of the amino acid histidine (for S. typhimurium) or tryptophan (for E. coli) required by the parent test strain.

Method

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using the Ames plate incorporation method at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors).   Two experiments were performed. The dose range for Experiment 1 was 1.5 to 5000 μg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. Toxicity was found to be excessive. The dose range was amended, following the results of Experiment 1, and was 1.5 to 1500 μg/plate. Up to seven test item dose levels were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the toxic limit of the test item.  

Results

There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in the first mutation test. Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, eitherwith or without metabolic activation in Experiment 2. A small, statistically significant increase in TA98 revertant colony frequency was observed in the presence of S9-mix at 150 μg/plate in the first mutation test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 150 μg/plate were within the in-house historical untreated/vehicle control range for the tester strain and only 1.6 times the concurrent vehicle control. Further statistically significant increases were also noted, however these responses were accompanied by weakened bacterial background lawns. Therefore the responses in this instance would be due to additional amino acid being available to the bacteria allowing the cells to undergo several additional cell divisions and presenting as non-revertant colonies.  

Expert conclusion

The test item is non-mutagenic under the conditions of this in vitro test.  

In Vitro Mammalian Chromosome Aberration Test–OECD No. 473

Principle

The in vitro chromosome aberration test identifies substances that cause structural chromosome aberrations in cultured mammalian cells. Structural aberrations may be either chromosomal or chromatid derived. Chromatid-type aberrations occur where breaks and re-joining affects only one of the sister-chromatids at any one locus. The majority of chemical mutagens induce chromatid aberrations type. Alternatively, chromosome-type aberrations occur where breaks and re-joining always affect both sister-chromatids at any one locus. Chromosome mutations are of greater concern since they may cause many genetic diseases in humans and experimental animals. Regardless of the result, it is important to note that the method is not intended to conclusively determine if the substance will cause chromosomal damage. It should be used as a screening method to decide if further genetic testing is required.  

Method

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study; i.e. in Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4 hour exposure period in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4 hour exposure with addition of S9 was repeated (using a 2% final S9 concentration) to investigate the reproducibility of the initial response, whilst in the absence of metabolic activation the exposure time was increased to 24 hours.  

Results

The test item was toxic and induced statistically significant increases in the frequency of cells with aberrations, in two of the 4 exposure groups tested. In Experiment 1, statistically significant increases in the frequency of aberrations were observed in the 4 hour exposure (20 hour expression period) with metabolic activation; and in Experiment 2 in the 24-hour continuous exposure group without metabolic activation.

 

Expert conclusion

Based on the results of thein vitrotest, that the substance is potentially clastogenic (i.e., an agent that gives rise to or induces disruption or breakages of chromosomes, leading to sections of the chromosome being deleted, added, or rearranged) in the intake animal. However, in evaluating the biological significance of thesein vitroresults, several factors must be considered:

-Screening purpose: This test is meant to be used as ascreen for possible mammalian mutagens and carcinogens. A positive result with thein vitrotest does not categorically indicate that the substance is a mutagen in a whole organism because it does not integrate the benefits of reparative possesses of the intact organism that would typically thwart chromosome damage.

- Potentially poor predictability:in vitrotests that have been followed-up within vivotesting have indicated that substances yielding a positive result in the in vitro studies are not always indicative of in vivo testing results [1].

- Cytotoxicity: Although secondary tests at lower doses suggested no indication of cytotoxicity, cytotoxicity leading to positive in vitro results [2] have resulted in apparent clastogenicity.

- False positives: The specificity ofin vitromammalian cell genotoxicity assays is low. They yield a high incidence of positive results that are not observed in animal genotoxicity tests and can be misleading or generate irrelevant positives that should be viewed with scrutiny [3].

- While the in vitro results suggest the substance appears to be clastogenic under the conditions of the test, it must only be considered to have the “potential to be clastogenic” within an intact organism. Follow on testing using in vivo methods were deemed to be therefore necessary to validate the in vitro chromosomal aberration results.

 

Overall expert conclusion for in vitro tests

The in vitro screening tests suggested the substance had the potential for clasotogenicity. Thus, a decision to evaluate genotoxicity usingin vivomethodologies was supported.

 

References

[1]Doktorova TY, Ates G, Vinken M, Vanhaecke T, Rogiers V. Way forward in case of a false positivein vitrogenotoxicity result for a cosmetic substance?. Toxicol in Vitro. 2014 Feb;28(1):54-9.

 

[2] Hilliard CA, Armstrong MJ, Bradt CI, Hill RB, Greenwood SK, Galloway SM. Chromosome aberrationsin vitrorelated to cytotoxicity of nonmutagenic chemicals and metabolic poisons. Environ Mol Mutagen. 1998;31(4):316-26. 

 

[3] Bryce SM, Bemis JC, Mereness JA, Spellman RA, Moss J, Dickinson D, Schuler MJ, Dertinger SD. Interpretingin vitromicronucleus positive results: simple biomarker matrix discriminates clastogens, aneugens, and misleading positive agents. Environ Mol Mutagen. 2014 Aug;55(7):542-55. 

  

In vivo Mammalian Erythrocyte Micronucleus Test – OECD No. 474

Principle

The purpose of the micronucleus test is to identify substances that cause cytogenetic damage in live animals which results in the formation of micronuclei containing either lagging chromosome fragments or whole chromosomes by damaging the mitotic apparatus of erythroblasts. The test is especially relevant for assessing genotoxicity becausein vivometabolism, pharmacokinetics and DNA repair processes are active in the whole animal and contribute to the responses.

 

Method

A range-finding test determined suitable dose levels of the test item and the potential for marked differences in the toxic response between the sexes. There was no marked gender difference in toxicity of the test item so the main test was performed using male mice. Animals were dosed via the intraperitoneal route in groups of seven at the 200 mg/kg (the maximum tolerated dose), 100 and 50 mg/kg and euthanized at 24 or 48 hours. The bone marrow was extracted, and smears were prepared and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei indicative of chromosomal damage. Negative controls (7 mice) were given a single oral dose of arachis oil. Positive controls (5 mice) were dosed orally with cyclophosphamide.

 

Results

In the intact organism, there were no statistically significant increases in the frequency of micronucleated PCEs in any of the test material dose groups when compared to the vehicle control group. The positive control group showed an increase in the incidence of micronucleated polychromatic erythrocytes confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide. No statistically significant decreases in the PCE/NCE ratio were observed in the 24 or 48-hour dosing groups when compared to the vehicle control group.

 

Expert conclusion

The results of thisin vivotest demonstrate that the substance is not cytogenic in the whole organism. This is especially significant given thein vitroscreening chromosomal aberration test results that suggested otherwise. Therefore, because thein vivotesting incorporates animal pharmacokinetics/pharmacodynamics and DNA repair processes that are active in the whole animal, it must be given greater emphasis in a weight of evidence evaluation of intrinsic and biologically relevant substance cytogenicity.

 

In vivo Mammalian Alkaline Comet Assay – OECD No. 489

Principle

Intrinsic mutagenic hazard of a substance must demonstrate DNA damage in intact animals. As noted above, thein vivomicronucleus test results indicate no damage to bone marrow cells of mice and add substantially to the weight of evidence that the substance is not inherently clastogenic. Using the Comet Assay, additionalin vivotesting was performed to verify the micronucleus results.

 

Thein vivo alkaline comet (single cell gel electrophoresis) assay detects DNA strand breaks in cells or nuclei isolated from multiple tissues of rats that have been exposed to the test substance. The test is designed to ensure that the animals have been exposed to the substance over a sufficient period of time such that it is distributed throughout the organism and is subject to metabolic processes that may lead to DNA damage as a fixed stable mutation or to repair resulting in no persistent effect.

 

Method

Male rats were used for the study since there was no marked difference in toxicity between the sexes in the above discussed previous mouse micronucleus study. Rats were administered arachis oil as a negative control and N-Nitroso-N-methylurea, in water, as a positive control. Rats subject to substance treatment were dosed twice within a 20 hour interval via gavage at 300, 600, and 1200 mg/kg in arachis oil. Rats from each dose level and controls were euthanized 24 hours following the initial treatment.

 

Samples of liver and glandular stomach were obtained from each animal. Cell suspensions were filtered through gauze prior to use for the comet slides. Once the cell suspensions had been obtained, approximately 30 μL was added to 270 μL of 0.5% low melting point (LMP) agarose, mixed thoroughly and 50 μL of this agarose/cell suspension mix was placed onto pre-coated slides. Two gels were placed on each slide, and 4 gels were prepared for each tissue (liver and glandular stomach).

 

The slides were placed into a lysing solution (pH 10) and refrigerated in the dark for at least 1 hour after which they were removed, rinsed with neutralization buffer, and placed into an electrophoresis bath for approximately 20 minutes to allow the DNA to unwind. DNA was subjected to electrophoresis at approximately 0.7 V/cm, 300 mA for approximately 20 minutes. The slides were removed and suspensions were neutralized, fixed in cold 100% methanol for 5 minutes and allowed to air dry.

 

Comet slides were hydrated, stained and placed onto the stage of a fluorescence microscope and scored for fluorescent comets. The primary endpoint was percentage DNA in the tail (percentage tail intensity). However, each slide was also assessed for the incidence of heavily damaged cells otherwise referred to as ‘hedgehog’ cells.

 

The median % DNA for each slide was determined the mean of the individual animal means was calculated to give a group mean. A statistical comparison was made between the appropriate vehicle control value and each individual dose level to determine if DNA damage was observed to be associated with exposure to the substance.

 

Results

There were no statistically significant increases in the group mean % tail intensity. This shows that the substance in vivo did not induce DNA damage in the cells of the glandular stomach (target tissue at the site of administration) or in the cells of the liver (target tissue at the primary site of metabolism after absorption).

 

Expert conclusion

The in vivo Comet assay did not indicate any DNA damage to the living organism. Therefore it should be considered to be non-genotoxic. These results corroborate those of the in vivo MNA and provide further evidence that the substance is not inherently genotoxic to the intact organism.

 

Overall expert conclusion for in vivo tests

Two in vivo studies were conducted and the test material was not found to be genotoxic. Furthermore, rats treated with test item for 90 consecutive days via oral gavage did not show neoplasia in various organs (see section 7.5 of this dossier). Therefore, the test material is unlikely to be genotoxic; no further genotoxicity testing is necessary or recommended.

Justification for classification or non-classification

In vivo studies conducted to clarify the results of in vitro screening tests concluded that the test material was not genotoxic. Classification in accordance with Regulation (EC) No 1272/2008 is therefore unnecessary.