Fatty acids, C12-18 (even numbered)-methyl esters, sulfonated, sodium salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

in vitro Bacterial reverse mutation assay: No significant increase in the numbers of revertant colonies was recorded with or without metabolic activation for any of the bacterial strains with any dose of test item. The test material was found to be non-mutagenic under the conditions of the test (OECD 471 and EU Method B.14).

in vitro Chromosome aberration test: The test item induced no toxicologically significant increases in the frequency of cells with aberrations or polyploid cells in the presence or absence of a liver enzyme metabolising system and was shown to be non-clastogenic to human cells in vitro (OECD 473 and EU Method B.10).

in vitro mammalian cell gene mutation test: No mutagenic effect of the test item was observed either in the presence or absence of metabolic activiation system under the conditions of this assay (OECD 476 and EU Method B.17).

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:

05 January 1994 to 17 January 1994

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)

Version / remarks:

Commission Directive 92/69/EEC

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 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced S9

Test concentrations with justification for top dose:

- Preliminary toxicity screen: 0, 312.5, 625, 1250, 2500, 5000 μg/plate.
- Experiment 1: 0, 8, 40, 200, 1000, 5000 μg/plate.
- Experiment 2 (TA100 and WP2uvrA): 0, 156.25, 312.5, 625, 1250, 2500, 5000 μg/plate.
- Experiment 2 (TA1535, TA98 and TA1537): 62.5, 125, 250, 500, 1000, 2000 μg/plate.

Vehicle / solvent:

Sterile distilled water

Negative solvent / vehicle controls:

yes

Remarks:

sterile distilled water

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

ENNG (2 μg/plate for WP2uvrA; 3 μg/plate for TA100; 5 μg/plate for TA1535 without metabolic activation)

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

9AA (80 μg/plate for TA1537 without metabolic activation)

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

4NQO (0.2 μg/plate for TA98 without metabolic activation)

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

2AA (2 μg/plate for TA1535 and 10 μg/plate for WP2uvrA with metabolic activation)

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

BP (5 μg/plate for TA100, TA1537 and TA98 with metabolic activation)

Details on test system and experimental conditions:

TESTER STRAINS
- Strains were obtained from the British Industrial Biological Research Association on 14 August 1987 and were stored at -196 °C in a Statebourne liquid nitrogen freezer (model SXR 34).
- Prior to being used, characterisation checks were performed to determine the amino acid requirement, presence of rfa, R factors, uvr mutation and the spontaneous reversion rate.
- In this assay, overnight subcultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited, lot number 35051940 11/97) and incubated at 37 °C for 10 hours.

PREPARATION OF TEST AND CONTROL MATERIALS
- Test item was accurately weighed (with an allowance made for purity) and dissolved in sterile distilled water. Appropriate dilutions were then made.
- The concentration, homogeneity and stability of the test material formulations were not determined by analysis.
- Negative and positive controls were used in parallel with the test material.

MICROSOMAL ENZYME FRACTION
- Lot number Aro S9/02/12/93 prepared on 02 December 1993 was obtained from the British Industrial Biological Research Association on 14 December 1993.
- The microsomal enzyme fraction was prepared from the livers of male Sprague-Dawley rats weighing approximately 200 g. The animals had received an i.p. injection of Aroclor 1254 at 500 mg/kg five days before S9 preparation.
- The S9 was stored at -196 °C in a Statebourne liquid nitrogen freezer (model SXR 34).

S9 MIX AND AGAR
- The S9 mix was prepared at 4 °C.
- 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) and sterile distilled water (14.5 mL).
- Top agar was prepared using 0.6 % Difco Bacto agar (lot 23050 12/97) and 0.5 % sodium chloride.
- For the Salmonella strains, 1.0 mM histidine/1.0 mM biotin solution (5 mL) was added to each 100 mL of top agar.
- For the Escherichia coli strain 1.0 mM tryptophan (5 mL) was added to each 100 mL of top agar.
- Base agar plates were prepared using 1.2 % Oxoid Agar Technical No 3 (lot number B224 70671 5/98) with Vogel-Bonner Medium E and 20 mg/mL D-glucose.

PRELIMINARY TOXICITY STUDY
- In order to select the appropriate dose levels for use in the main study, a preliminary test was performed to determine the toxicity of the test material to the tester organisms.
- TA100 or WP2uvrA bacterial suspension (0.1 mL), molten trace histidine or tryptophan supplemented media (2 mL), test solution (0.1 mL) and phosphate buffer (0.5 mL) were overlayed onto sterile plates of Vogel-Bonner minimal agar (30 mL/plate).
- Five doses of the test material and a solvent control were tested in duplicate.
- The plates were scored for revertant colonies and examined for thinning of the background lawn after 48 hours incubation at 37 °C.

MUTATION STUDY - EXPERIMENT 1
- Five concentrations of the test material were assayed in triplicate against each tester strain using the direct plate incorporation method in accordance with the standard methods for mutagenicity tests using bacteria.
- Plates were incubated at 37 °C for 48 hours and the number of revertant colonies counted.

TEST MATERIAL AND NEGATIVE CONTROLS
- Aliquots (0.1 mL) of one of the bacterial suspensions were dispensed into sets of sterile test tubes followed by molten trace histidine or tryptophan supplemented top agar (2.0 mL) at 45 °C. These sets comprised two test tubes for each bacterial tester strain.
Appropriately diluted test material (0.1 mL) or negative control solution was also added to each of the two tubes, followed by S9 liver microsome mix (0.5 mL) or pH 7.4 buffer (0.5 mL).
- The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner agar plates (one tube per plate).
- The procedure was repeated in triplicate for each bacterial strain and each concentration of test material.

POSITIVE CONTROLS - WITHOUT ACTIVATION
- One of the positive control solutions (ENNG, 9AA or 4NQO) was added to a test tube containing molten trace histidine or tryptophan supplemented top agar (2.0 mL) and the appropriate bacterial suspension (0.1 mL).
- Finally, pH 7.4 buffer (0.5 mL) was added to the tube. The contents were then mixed and poured onto agar plates.
- The procedure was then repeated in triplicate for each of the positive controls.

POSITIVE CONTROLS - WITH ACTIVATION
- 2AA or BP solution (0.1 mL) was added to a test tube containing molten trace histidine or tryptophan supplemented top agar (2.0 mL) and the appropriate bacterial suspension (0.1 mL).
- Finally, S9 mix (0.5 mL) was added to the tube. The contents were then mixed and poured onto agar plates.
- The procedure was then repeated in triplicate for each tester strain.

EXPERIMENT 2
- The second experiment was performed in triplicate using fresh bacterial cultures, six concentrations of test material for each strain and control solutions.

Evaluation criteria:

INTERPRETATION OF RESULTS
- For a substance to be considered positive in this test system, it should have induced a dose-related and statistically significant increase in mutation rate (of at least twice the spontaneous reversion rate) in one or more strains of bacteria in the presence and/or absence of the S9 microsomal enzymes in both experiments at sub-toxic dose levels.
- If the two experiments give conflicting results then a third experiment may be used to confirm the correct response.
- To be considered negative the number of induced revertants should be less than twofold at each dose level employed, the intervals of which should be between 2 and 5 fold and extend to the limits imposed by toxicity or solubility or up to the maximum recommended dose of 5000 μg/plate. In this investigation the limiting factor was both toxicity and maximum recommended dose.

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:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY STUDY
- Test material was non-toxic in the strains of bacteria used (TA100 and WP2uvrA).
- Mean numbers of revertant colonies for the toxicity assay are shown in the table below.

MUTATION STUDY
- Results of the checks for characteristics, viability and spontaneous reversion rate for each tester strain were all found to be satisfactory.
- Individual plate counts for Experiment 1 are shown in Tables 1 and 2 (attached) together with the mean number of revertant colonies obtained for each tester strain following incubation with test material (with and without metabolic activation).
- Individual plate counts for Experiment 2 are shown in Tables 3 and 4 (attached) together with the mean number of revertant colonies obtained for each tester strain following incubation with test material (with and without metabolic activation).
- Results are expressed graphically in Appendix II (attached).
- Results for positive controls are shown in Tables 1 to 4 (attached).
- Test material caused a reduction in the growth of the bacterial lawn at and above 2000 μg/plate in Salmonella strains TA1535, TA98 and TA1537 both with and without metabolic activation. The test item was therefore investigated up to either the maximum recommended dose level of 5000 μg/plate or the toxic limit of a particular bacterial strain. A precipitate was observed at the maximum dose but this did not interfere with scoring of revertant colonies.
- No significant increases in the numbers of revertant colonies of bacteria were recorded at any dose level for any of the strains of bacteria used (with or without metabolic activation).
- The positive control substances all produced marked increases in the number of revertant colonies and the activity of the S9 fraction was found to be satisfactory.

Mean numbers of revertant colonies in the preliminary toxicity study
Stain	Dose (μg/plate)
	0	312.5	625	1250	2500	5000
TA100	110.0	140.0	123.0	123.5	119.5	102.5
WP2uvrA	36.5	27.0	38.5	31.5	34.5	26.5 (P)
(P) = precipitate

Conclusions:

No significant increase in the numbers of revertant colonies was recorded with or without metabolic activation for any of the bacterial strains with any dose of test item. The test material was found to be non-mutagenic under the conditions of the test.

Executive summary:

In accordance with OECD 471 and EU Method B.14, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 plus Escherichia coli strain WP2uvrA were treated with test material by the Ames plate incorporation method at five and six dose levels. The investigation was conducted in triplicate with and without addition of rat liver homogenate metabolising system at 10 % in standard co-factors. The dose range was determined in a preliminary toxicity assay and was 8 to 5000 μg/plate in the first experiment. The experiment was repeated on a separate day using different cultures of the bacterial strains and fresh chemical formulations. In this case, various dose ranges of test material were used to allow for differential toxicity to the bacterial strains tested.

The solvent (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All positive control chemicals produced marked increases in the numbers of revertant colonies, both with and without the metabolising system. The test item caused a reduction in the growth of the bacterial lawn at and above 2000 μg/plate in Salmonella strains TA1535, TA98 and TA1537 with and without metabolic activation. The test item was therefore tested up to either the maximum recommended dose level of 5000 μg/plate or the toxic limit of a particular bacterial strain. A precipitate was observed at the maximum dose but did not interfere with the scoring of revertant colonies.

No significant increase in the numbers of revertant colonies was recorded with or without metabolic activation for any of the bacterial strains with any dose of test item. The test material was found to be non-mutagenic under the conditions of the test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 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)

Version / remarks:

Commission Directive 92/69/EEC

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: clastogenisity

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Cells were grown in Eagle's minimal essential medium (supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin, amphotericin B and 15 % foetal calf serum) at 37 °C with 5 % CO2 in air. The lymphocytes in fresh heparinised whole blood were stimulated to divide by the addition of phytohaemaglutinin (PHA) at 90 μg/mL final concentration.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

EXPERIMENT 1
0, 39, 78.1, 156.25, 312.5, 625, 1250, 2500 and 5000 μg/mL in cultures with and without S9

EXPERIMENT 2
0, 78.1, 117.2, 156.25, 312.5, 468.75, 625 and 1250 μg/mL for 20-hour treatment with and without S9
0, 117.2, 156.25, 625 and 1250 μg/mL for 44-hour treatment with and without S9

Vehicle / solvent:

Sterile distilled water

Negative solvent / vehicle controls:

yes

Remarks:

sterile distilled water

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

EMS (Sigma batch 32H0829; 500 μg/mL dissolved in dimethyl sulphoxide for cultures in the absence of metabolising enzymes)

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

CP (Sigma batch 72H0088; 25 μg/mL dissolved in culture medium without serum for cultures where S9 was included)

Details on test system and experimental conditions:

CELLS
- Sufficient whole blood was drawn from the peripheral circulation of volunteers who had been previously screened for suitability.
- The volunteers had not been exposed to high levels of radiation, hazardous chemicals and had not knowingly suffered from a viral infection.
- Cell cycle time for the lymphocytes from the donors used in the study was determined using bromodeoxyuridine (BrdU) incorporation to assess the number of first, second and third division metaphase cells and calculate the average generation time (AGT).
- Using the batch of foetal calf serum in available at the time, the donor used in the study had AGT values in the range 11.5 to 14 hours.

PREPARATION OF TEST AND CONTROL MATERIALS
- Test material was accurately weighed and dissolved in sterile distilled water. Appropriate serial dilutions were then made.
- Analysis of the test material preparations for homogeneity, stability and concentration is not a requirement of the test guideline and was not performed.

MICROSOMAL ENZYME FRACTION
- Lot Aro S9/02/12/93 (prepared 02 December 1993) was obtained from the British Industrial Biological Research Association on 14 December 1993 and used in Experiment 1.
- Lot Aro S9/03/02/94 (prepared 03 February 1994) was obtained from the British Industrial Biological Research Association on 22 February 1994 and used in Experiment 2.
- Microsomal enzyme fractions were prepared from the livers of male Sprague-Dawley rats weighing 200 g. The animals had received a single i.p. injection of Aroclor 1254 at 500 mg/kg five days before the S9 preparation.
- The S9 was stored at -196 °C in a Statebourne liquid nitrogen freezer (model SXR 34).

CULTURE CONDITIONS - EXPERIMENT 1
- Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing components in bulk.
- When dispensed into sterile plastic flasks, each dose level contained 8.05 mL MEM, 15 % (FCS) for cultures in the absence of S9; 9.05 mL MEM, 15 % (FCS) for cultures in the presence of S9 and for positive control cultures in the absence of S9; 0.1 mL Li-heparin; 0.1 mL phytohaemagglutinin-M; 0.75 mL heparinised whole blood.
- After approximately 48 hours incubation at 37 °C and 5 % CO2 in humidified air, the cells of the with-S9 cultures were centrifuged for 5 minutes and all but 1 mL of the culture medium was removed, reserved, and replaced with 7.0 mL of MEM (7.9 mL added to positive control cultures).
- Appropriate solution of vehicle (1.0 mL) or test item was added to each culture.
- Positive control cultures were dosed at 0.1 mL volumes.
- Final concentrations of test item were 5000, 2500, 1250, 625, 312.5, 156.25, 78.1 and 39 μg/mL in cultures with and without S9.
- 10 % S9 (1 mL in standard co-factors) was added to the with-S9 cultures.
- All cultures were then returned to the incubator.
- After four hours of treatment at 37 °C, the with-S9 cultures were centrifuged for five minutes and treatment medium was removed by suction before being 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.
- Cells were then re-incubated for a further 16 hours.

CULTURE CONDITIONS - EXPERIMENT 2
- Culture conditions were as for the previous experiment except that further cultures of lymphocytes were harvested 44 hours after the initiation of treatment in addition to the 20-hour harvest in Experiment 1.
- Positive controls were evaluated only in the 20-hour harvest cultures.

CELL HARVEST
- Mitosis was arrested by addition of demecolcine (0.1 μg/mL) two hours before the required harvest time.
- After incubation with demecolcine, the cells were centrifuged, culture medium was drawn off and discarded, and the cells were resuspended in 8 mL of 0.075 M KCl.
- After 10 minutes (including 5 minutes centrifugation), all but approximately 2.0 mL of hypotonic solution was drawn off and discarded.
- The cells were resuspended and then fixed by dropping the KCl suspension into approximately 3.0 mL of fresh methanol/glacial acetic acid (3:1 v/v).
- The fixative was changed at least twice and the cells were stored at 4 °C for at least four hours to ensure complete fixation.

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

STAINING
- When slides were dry, they were stained in 5 % Gurrs Giemsa R66 for five minutes.
- Slides were then rinsed, dried and mounted in Depex mounting medium.

CODING
- After checking that the slide preparations were of good quality, the slides were coded using a code from a computerised random number generator.

Evaluation criteria:

SCORING OF CHROMOSOME DAMAGE
- Where possible the first 100 consecutive well-spread metaphases from each culture were counted.
- If the cell had 46 or more 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.
- Details of the classification of chromosome aberrations and the evaluation criteria applied to the test data are given in Appendix 1 (attached).
- All chromosome aberrations were checked by a senior cytogeneticist prior to decoding the slides.

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

Statistics:

- The frequency of cells with aberrations (including and excluding gaps) and the frequency of polyploid cells was compared with the concurrent vehicle control value using Fisher's Exact test or Chi-squared test.

Key result

Species / strain:

other: human

Remarks:

lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

EXPERIMENT 1
- Doses selected for scoring cells with aberrations in the without-S9 group were 39, 78.1 and 156.25 μg/mL.
- The limiting factor for dose selection was toxicity as there were no scorable metaphases at 312.5 μg/mL.
- Doses selected for the with-S9 group were 156.25, 312.5 and 625 μg/mL. There were no scorable metaphases at 1250 μg/mL.
- Vehicle control cultures had a high frequency of cells with chromosome aberrations without metabolic activation treatment and a low frequency of cells with chromosome aberrations when metabolic activation was used (see Table 4, attached).
- Without S9, the highest frequency of cells with aberrations seen in the two vehicle control groups was 9.0 % cells with aberrations including gaps and 4.0 % cells with aberrations excluding gaps.
- Whilst the vehicle control without S9 aberration frequency was outside the historical range, it was considered that the response was due more to "chance" selection of metaphases than to treatment and was therefore spurious.
- No effect was seen in the test material cultures and no effect was seen in further investigations of the donor's blood.
- Positive control treatments gave significant increases in the frequency of cells with aberrations (see Table 4, attached). This indicated that the metabolic activation system was satisfactory and that the test method was operating as expected.
- The test material was seen to induce a statistically significant increase in the frequency of cells with aberrations in the presence of S9 (see Table 4, attached). The observed increases were at dose levels of 312.5 and 625 μg/mL but was not seen in both of the duplicate cultures, was not reproducible, and was only significant including gaps. The result was therefore considered to be of no toxicological significance.
- Test material did not induce a significant increase in the numbers of polyploid cells at any dose level in either of the treatment cases (see Table 7, attached).

EXPERIMENT 2
- Doses were selected on the basis of data from Experiment 1 and comprised a narrower dose range.
- Doses selected for evaluation were 78.1, 117.2 and 156.25 μg/mL for 20-hour treatment without S9.
- Doses selected for evaluation were 312.5, 468.75 and 625 μg/mL for 20-hour treatment with S9.
- The dose selected for evaluation was 117.2 μg/mL for 44-hour treatment without S9.
- The dose selected for evaluation was 625 μg/mL for 44-hour treatment with S9.
- All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the historical range (see Tables 5 and 6, attached).
- Positive control treatments gave significant increases in the frequency of cells with aberrations (see Table 5, attached) indicating that the metabolic activation system was satisfactory and that the test system was operating as expected.
- Test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in the 20 and 44-hour treatment groups in the presence or absence of S9-mix (see Tables 5 and 6, attached).
- Test material did not induce a significant increase in the numbers of polyploid cells at any dose level in any of the treatment cases (see Table 7, attached).

Conclusions:

The test item induced no toxicologically significant increases in the frequency of cells with aberrations or polyploid cells in the presence or absence of a liver enzyme metabolising system and was shown to be non-clastogenic to human cells in vitro.

Executive summary:

Human lymphocytes treated with test material were evaluated for chromosome aberrations in accordance with OECD 473 and EU Method B.10 at three dose levels, in duplicate, together with vehicle and positive controls. Four treatment conditions were used (4 hours exposure with the addition of an induced rat liver homogenate metabolising system at 10 % in standard co-factors with cell harvest after 16 and 40 expression periods and a 20 and 44 hour continuous exposure in the absence of activation). In Experiment 1, the dose range for evaluation was selected from a series of eight dose levels on the basis of toxicity.

Most vehicle (solvent) controls gave frequencies of cells with aberrations within the current historical range for human lymphocytes. All the positive control treatments gave significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test item induced no toxicologically significant increases in the frequency of cells with aberrations or polyploid cells in the presence or absence of a liver enzyme metabolising system and was shown to be non-clastogenic to human cells in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23rd February - 21st August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro mammalian cell gene mutation

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

Sub-line (K1)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: American Type Culture Collection (ATCC)
- Suitability of cells:
- Cell cycle length, doubling time or proliferation index:
- Sex, age and number of blood donors if applicable:
- Whether whole blood or separated lymphocytes were used if applicable:
- Number of passages if applicable:
- Methods for maintenance in cell culture if applicable:
- Modal number of chromosomes:
- Normal (negative control) cell cycle time:

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:F12-10 medium, 0.3% CO2 in air
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically 'cleansed' against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and beta-naphthoflavone-induced rat liver S9

Test concentrations with justification for top dose:

Treatment concentrations for the mutation assays of the main tests were selected based on the results of a preliminary toxicity test as follows:
Assay 1
5-hour treatment in the presence of S9-mix:
120, 110, 100, 90, 80, 70, 60, 30 and 15 µg/mL
5-hour treatment in the absence of S9-mix:
60, 55, 50, 45, 40, 35, 17.5, 7 and 5 µg/mL
Assay 2
5-hour treatment in the presence of S9-mix:
500, 250, 125, 93.75, 62.5, 31.25, 15.625 and 7.813 µg/mL
5-hour treatment in the absence of S9-mix:
500, 250, 125, 93.75, 62.5, 46.875, 31.25, 15.625, 7.813 and 3.906 µg/mL
24-hour treatment in the absence of S9-mix:
500, 250, 125, 93.75, 62.5, 31.25, 15.625 and 7.813 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: Substance is soluble in water

Untreated negative controls:

yes

Remarks:

Distilled water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO - solvent for DMBA

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation

Untreated negative controls:

yes

Remarks:

Distilled water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO - solvent for MES

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 hours
- Exposure duration: Assay 1 - 5-hour with or without S9-mix; Assay 2 - 5-hour with or without S9-mix; 24-hour without S9-mix
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: 5

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: After the growing or selection period, the culture medium was removed and colonies were fixed for 5 minutes with methanol. After fixation, colonies were stained using 10% Giemsa solution (diluted with distilled water) for 30 minutes, dried and manually counted.

NUMBER OF CELLS EVALUATED: 2E06 cells: 5 plates at 4E05 cells/plate

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
- Any supplementary information relevant to cytotoxicity: Other parameters measuring cytotoxicity like relative survival after treatment (comparing the cell number determined after treatment to the vehicle (solvent) control) or relative total growth (comparing the growth rate during the entire expression period to the vehicle (solvent) control) were also calculated.

Evaluation criteria:

The test item was considered to be mutagenic in this assay if the following criteria are met:
1. The assay is valid.
2. The mutant frequency at one or more doses is significantly greater than that of the relevant negative (vehicle) control (p<0.05).
3. Increase of the mutant frequency is reproducible.
4. There is a dose-response relationship.
Results which only partially met the criteria were dealt with on a case-by-case basis (historical control data of untreated control samples was taken into consideration if necessary). Similarly, positive responses seen only at high levels of cytotoxicity required careful interpretation when assessing their biological significance. Indeed, extreme caution was exercised with positive results obtained at levels of survival lower than 10%.
According to the relevant OECD guideline, the biological relevance of the results was considered first, statistical significance was not the only determination factor for a positive response.

Statistics:

The homogeneity of variance between groups was checked by Bartlett`s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was made. If the obtained result were significant, Duncan’s Multiple Range test was used to assess the significance of inter-group differences. Significant results with inter-group comparisons were further compared using Kruskal-Wallis and Mann-Whitney U-tests.
The mutation frequencies were statistically analyzed. Statistical evaluation of data was performed with the SPSS PC+4.0 statistical program package (SPSS Hungary Ltd., Budapest, Hungary). The heterogeneity of variance between groups was checked by Bartlett`s test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan’s Multiple Range test was used to assess the significance of inter-group differences.
Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorow-Smirnow test. In the case of not normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If a positive result was detected, the inter-group comparisons were performed using Mann-Whitney U-test. Data also were checked for a trend in mutation frequency with treatment dose using Microsoft Excel 2010 software (R-squared values were calculated for the log concentration versus the mutation frequency).

Species / strain:

Chinese hamster Ovary (CHO)

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There were no large changes in pH after treatment in any cases.
- Effects of osmolality: There were no large changes in osmolality after treatment in any cases.
- Evaporation from medium: Not applicable
- Water solubility: No
- Precipitation: None observed
- Definition of acceptable cells for analysis:
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES:

5-h +/-S9 & 24-h -S9: Cytotoxicity from 125 μg/mL (0% survival after day 1 of treatment)

Results of range finding study are attached.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive controls:
DMBA (5-h, +S9): mean = 905.2; SD = 562,7; range = 141.2-2119.4; n = 27
EMS (5-h, -S9): mean = 445.6; SD = 118.6; range = 239.6-636.6; n = 13
EMS (24-h, -S9): mean = 1176.6; SD = 610.9; range = 363.1-2449.8; n = 14
- Untreated control:
5-h, +S9: mean = 18.3; SD = 15.1; range = 5.1-64.1; n = 27
5-h, -S9: mean = 20.7; SD = 16.4; range = 5.5-55.5; n = 13
24-h, -S9: mean = 19.0; SD = 17.2; range = 3.3-58.0; n = 14
- DMSO controls:
5-h, +S9: mean = 21.8; SD = 15.9; range = 5.4-57.3; n = 29
5-h, -S9: mean = 18.9; SD = 11.6; range = 6.5-47.4; n = 13
24-h, -S9: mean = 18.4; SD = 14.4; range = 6.8-48.5; n = 14
- Distilled water / Water based vehicle control:
5-h, +S9: mean = 11.5; SD = 3.8; range = 6.1-15.8; n = 6
5-h, -S9: mean = 9.1; SD = 3.4; range = 5.2-11.6; n = 3
24-h, -S9: mean = 15.5; SD = 5.6; range = 9.2-20.1; n = 3

Assay 1:

5-h, +S9 mix: Marked cytotoxicity - highest concentration of 120 μg/mL showed a relative survival of 4.4% and 89% after treatment and on the survival plates, respectively. An evaluation was made using all 9 concentrations. Slight, statistically significant increase (at p<0.05 level) was observed in this experiment at 90, 80 and 60 μg/mL concentrations although the observed values were within the general historical control

range. Furthermore, the observed mutant frequencies (6.8, 6.4 and 5.0 x 10-6) were within the expected range of the negative control samples according to the relevant OECD guideline (expected range: 5-20 x 10-6). No clear dose response to the treatment was observed (a trend analysis showed no effect of treatment).

5-h, -S9 mix: Marked cytotoxicity - highest concentration of 60 μg/mL showed a relative survival of 9.4% and 99% after treatment and on the survival plates, respectively. An evaluation was made using data of all nine concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment).

Assay 2:

5-h, +S9 mix: Excessive cytotoxicity was observed at 500 and 250 μg/mL concentration with no surviving cells detected after the treatment. An evaluation was made using data of the following concentration of 125 μg/mL (relative survival of 0.8% and 65% after treatment and on the survival plates, respectively) and the next five concentrations (a total of six concentrations). No statistically significant increases in the numerical value of the mutation frequency compared to the negative (vehicle) control were observed at any examined concentrations and there was no dose response to the treatment (a trend analysis showed no effect of treatment).

5-h, -S9 mix: Excessive cytotoxicity was observed at 500, 250, 125, 93.75 and 62.5 μg/mL concentration with no surviving cells detected after the treatment. An evaluation was made using data of the following concentration of 46.875 μg/mL (relative survival of 6.8% and 66% after treatment and on the survival plates, respectively) and the next four concentrations (a total of five concentrations). No statistically significant increases in the numerical value of the mutation frequency compared to the negative (vehicle) control were observed at any examined concentrations and there was no dose response to the treatment (a trend

analysis showed no effect of treatment).

24-h, -S9 mix: Excessive cytotoxicity was observed at 500, 250 and 125 μg/mL concentration with no surviving cells detected after the treatment. An evaluation was made using data of the following concentration of 93.75 μg/mL (relative survival of 7.9% and 77% after treatment and on the survival plates, respectively) and the next four concentrations (a total of five concentrations). No statistically significant increases in the numerical value of the mutation frequency compared to the negative (vehicle) control were observed at any examined concentrations and there was no dose response to the treatment (a trend analysis showed no effect of treatment).

The other sporadic, statistically non-significant differences were examined for consistency; none of them were repeatable when comparing Assay 1 and Assay 2. Furthermore, all the observed mutation frequency values were within the general historical control range. Together with the lack of correlation with dose level, this confirms that there were no biologically significant differences between treated samples

and negative (vehicle) controls.

Results tables are attached.

Conclusions:

No mutagenic effect of the test item was observed either in the presence or absence of metabolic activation system under the conditions of this HPRT assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo mouse micronucleus test: The test material was considered to be non-genotoxic under the stated experimental conditions (OECD 474 and EU Method B.12).

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 January 1994 to 25 February 1994

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)

Version / remarks:

Commission Directive 92/69/EEC

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

Sex:

female

Details on test animals or test system and environmental conditions:

ANIMALS AND ANIMAL HUSBANDRY
- Sufficient male and female albino CDI strain mice were supplied by Charles River (UK) Ltd, Manston, Kent, UK.
- At the start of the main study animals weighed 22-29 g (males) or 20-24 g (females) and were approximately five to eight weeks old.
- After a minimum acclimatisation period of five days, animals were selected at random and given a unique number within the study by ear punching and a number written on a colour coded cage card.
- Animals were housed in groups of up to five by sex in solid-floor polypropylene cages with woodflakes bedding.
- Free access to mains drinking water and food (Rat and Mouse Expanded Diet No 1, Special Diets Services, Limited, Witham, Essex, UK) was allowed throughout the study.
- The animal room was maintained at a temperature of 19 to 21 °C with relative humidity of 44 to 46 %.
- Rate of air exchange was approximately 15 changes per hour.
- Lighting was controlled by a time switch to give 12 hours light and 12 hours darkness.

Route of administration:

intraperitoneal

Vehicle:

Sterile distilled water supplied by Steripak and stored at room temperature.

Details on exposure:

RANGE-FINDING TOXICITY STUDY
- A range-finding study was performed to determine a suitable dose level and route of administration for the micronucleus study.
- The dose level selected should ideally be the maximum tolerated dose or that which produces some evidence of cytotoxicity up to a maximum recommended dose of 2000 mg/kg.
- Intraperitoneal dose levels were 50, 100, 200, 500, 1000 and 2000 mg/kg (one male and one female per dose level with administration via a hypodermic needle attached to a graduated syringe).
- Gavage dose levels were 500, 1000 and 2000 mg/kg (one male and one female per dose level with administration via a metal cannula).
- 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 per day for two days.
- Any deaths and evidence of overt toxicity were recorded at each observation.
- No necropsies were performed.

MICRONUCLEUS STUDY
- Groups of ten mice (five males and five females) were dosed once only via the intraperitoneal route with 50, 100 or 200 mg/kg test material.
- Dose groups, kill time and associated information are provided in the table below.
- Animals were killed by cervical dislocation.
- All animals were observed for signs of overt toxicity and death one hour after dosing and then once daily and immediately prior to sacrifice.

Duration of treatment / exposure:

24 or 48 hours

Frequency of treatment:

Single dose

Post exposure period:

Not applicable

Dose / conc.:

50 other: mg/kg

Remarks:

Single dose; intraperitoneal route

Dose / conc.:

100 other: mg/kg

Remarks:

Single dose; intraperitoneal route

Dose / conc.:

200 other: mg/kg

Remarks:

Single dose; intraperitoneal route

Control animals:

yes, concurrent vehicle

Positive control(s):

- Cyclophosphamide was supplied by the Sigma Chemical Company and stored at 0-5 °C.
- The positive control material was freshly prepared when required as a solution of appropriate concentration in distilled water.
- The concentration, homogeneity and stability of the positive control material and its preparation were not determined by analysis.

Tissues and cell types examined:

One femur was dissected from each animal immediately following sacrifice.

Details of tissue and slide preparation:

SLIDE PREPARATION
- Femurs were aspirated with foetal calf serum and bone marrow smears were prepared following centrifugation and resuspension.
- Smears were air-dried, fixed in absolute methanol and stained in May-Grünwald/Giesma.

Evaluation criteria:

- Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification.
- The incidence of micronucleated cells per 1000 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, and have a sharp contour with even staining.
- The number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes were counted. These cells were also scored for incidence of micronuclei.
- The ratio of polychromatic to normochromatic erythrocytes was calculated with appropriate group mean values for males, females and all animals.

Statistics:

- A comparison was made between the number of micronucleated polychromatic erythrocytes occuring in each of the test material groups and the number occuring in the corresponding vehicle control group.
- A positive mutagenic response is demonstrated when a statistically significant increase in the number of micronucleated polychromatic erythrocytes is observed for either the 24 or 48-hour kill times when compared to the corresponding vehicle control group.
- If these criteria are not demonstrated, the test material is considered to be non-genotoxic under the conditions of the test.
- A positive response for bone marrow toxicity is demonstrated when the dose group mean polychromatic to normochromatic ratio is shown to be statistically significant from the concurrent vehicle control group.
- All data were statistically analysed using appropriate methods as recommended by the UKEMS sub-committee on guidelines for mutagenicity testing, Report, Part III (1989).
- Data was analysed following a square route of (x+1) transformation using Student's t-test (two tailed) and any significant results were confirmed using one-way analysis of variance.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING STUDY
- Mortality data from the range-finding study are presented in the attached table.
- No premature animal deaths took place after dosing with test material via the oral route.
- Clinical signs oversed in animals dosed with test item via the intraperitoneal route were lethargy, decreased respiratory rate, laboured respiration, ptosis and hunched posture.
- Premature animal deaths occurred in animals dosed at and above 500 mg/kg via the intraperitoneal route. Clinical observations seen only at 500 mg/kg were ataxia, pallor of the extremities and walking with splayed gait.
- Based on these results, the main study was conducted using the intraperitoneal route with 200 mg/kg as the maximum tolerated dose (MTD) and 100 mg/kg and 50 mg/kg as the other dose levels.

MICRONUCLEUS STUDY
- One premature animal death was seen at 48 hours in the 200 mg/kg test item group.
- Clinical signs observed in animals dosed with test material were hunched posture, lethargy, decreased respiratory rate, laboured respiration, pilo-erection, ptosis and tiptoe gait.
- Lethargy, decreased respiratory rate and laboured respiration were observed one hour after dosing at 50 mg/kg and 100 mg/kg.
- With the exception of one animal in the 100 mg/kg group no clinical signs were observed at 24 hours after dosing.
- In the 200 mg/kg group, clinical signs were seen at one hour, 24 hours and 48 hours after dosing.

EVALUATION OF BONE MARROW SLIDES
- Results are summarised in Table 1 (attached).
- Individual and group mean data are presented in Tables 2 to 8 (attached).
- There was no statistically significant increase in the frequency of micronucleated PCEs in any of the test material dose groups when compared to their concurrent vehicle control groups.
- There was no significant change in the PCE/NCE ratio in any of the test material dose groups when compared to their concurrent vehicle control groups.
- The presence of clinical observations demonstrates that systemic absorption of test material had taken place.

EVALUATION OF BONE MARROW SLIDES
- Test material was found not to produce a significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice under the conditions of the test.

Conclusions:

The test material was considered to be non-genotoxic under the stated experimental conditions.

Executive summary:

A study was performed in accordance with OECD 474 and EU Method B.12 to assess the potential of the test item to produce damage to chromosomes and/or aneuploidy when administered via the intraperitoneal route to mice. Following a preliminary range-finding study to confirm the toxicity of the test material and route of administration, the micronucleus study was conducted using the intrperitoneal route in groups of ten mice (five male and five female) at 50 mg/kg, 100 mg/kg and the maximum tolerated dose of 200 mg/kg. Animals were killed 24 or 48 hours later, the bone marrow was extracted and smear preparations were made and stained. Polychromatic and normochromatic erythrocytes were scored for the presence of micronuclei. Further groups of mice were given a single intraperitoneal dose of distilled water to serve as vehicle controls or dosed orally with cyclophosphamide to serve as positive controls.

There was no evidence of an increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with test material when compared to the concurrent vehicle control group. No significnat change in the PCE/NCE ratio was observed after dosing with test item although the presence of clinical observations indicated that systemic absorption had occurred. The positive control material produced a marked increase in the frequency of micronucleated polychromatic erythrocytes. The test material was considered to be non-genotoxic under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

in vitro bacterial reverse mutation assay: In accordance with OECD 471 and EU Method B.14, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 plus Escherichia coli strain WP2uvrA were treated with test material by the Ames plate incorporation method at five and six dose levels. The investigation was conducted in triplicate with and without addition of rat liver homogenate metabolising system at 10 % in standard co-factors. The dose range was determined in a preliminary toxicity assay and was 8 to 5000μg/plate in the first experiment. The experiment was repeated on a separate day using different cultures of the bacterial strains and fresh chemical formulations. In this case, various dose ranges of test material were used to allow for differential toxicity to the bacterial strains tested. The solvent (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All positive control chemicals produced marked increases in the numbers of revertant colonies, both with and without the metabolising system. The test item caused a reduction in the growth of the bacterial lawn at and above 2000μg/plate in Salmonella strains TA1535, TA98 and TA1537 with and without metabolic activation. The test item was therefore tested up to either the maximum recommended dose level of 5000μg/plate or the toxic limit of a particular bacterial strain. A precipitate was observed at the maximum dose but did not interfere with the scoring of revertant colonies. No significant increase in the numbers of revertant colonies was recorded with or without metabolic activation for any of the bacterial strains with any dose of test item. The test material was found to be non-mutagenic under the conditions of the test.

in vitro chromosome aberration test: Human lymphocytes treated with test material were evaluated for chromosome aberrations in accordance with OECD 473 and EU Method B.10 at three dose levels, in duplicate, together with vehicle and positive controls. Four treatment conditions were used (4 hours exposure with the addition of an induced rat liver homogenate metabolising system at 10 % in standard co-factors with cell harvest after 16 and 40 expression periods and a 20 and 44 hour continuous exposure in the absence of activation). In Experiment 1, the dose range for evaluation was selected from a series of eight dose levels on the basis of toxicity. Most vehicle (solvent) controls gave frequencies of cells with aberrations within the current historical range for human lymphocytes. All the positive control treatments gave significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test item induced no toxicologically significant increases in the frequency of cells with aberrations or polyploid cells in the presence or absence of a liver enzyme metabolising system and was shown to be non-clastogenic to human cells in vitro.

in vitro mammalian cell gene mutation assay: An in vitro mammalian cell assay was performed in accordance with OECD 476 and EC Method B17in CHO K1 Chinese hamster ovary cells at thehprtlocus to evaluate the potential of the test item to cause gene mutation. Treatments were carried out for 5 hours with and without metabolic activation (±S9-mix) and for 24 hours without metabolic activation (-S9-mix). Distilled water was used as the vehicle (solvent) of the test item in this study. Treatment concentrations for the mutation assays of the main tests were selected based on the results of a preliminary toxicity test.

 

In the main assays, a measurement of the survival, viability and mutagenicity was determined. In Assays 1 and 2, no insolubility was detected in the final treatment medium at the end of the treatment with or without metabolic activation and there were no large changes in pH and osmolality after treatment in any cases.

 

In Assay 1, marked cytotoxicity was observed at the highest concentration (120 μg/mL +S9, of 60 μg/mL -S9). In the +S9 experiment, a slight, statistically significant increase (at p<0.05 level) was observed at 90, 80 and 60 μg/mL concentrations, although the observed values were within the general historical control range. No clear dose response to the treatment was observed (a trend analysis showed no effect of treatment). In the -S9 experiment no statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment).

 

In Assay 2, in the absence of S9-mix (5-hour treatment), excessive cytotoxicity was observed at 62.5 μg/mL and above with no surviving cells detected after the treatment. In the evaluated concentrations, no statistically significant increases in the numerical value of the mutation frequency compared to the negative (vehicle) control were observed at any examined concentrations and there was no dose response to the treatment (a trend analysis showed no effect of treatment).

 

In the presence of S9-mix (5-hour treatment), excessive cytotoxicity was observed at 250 μg/mL and above with no surviving cells detected after the treatment. In the evaluated concentrations, no statistically significant increases in the numerical value of the mutation frequency compared to the negative (vehicle) control were observed at any examined concentrations and there was no dose response to the treatment (a trend analysis showed no effect of treatment).

 

In Assay 2, in the absence of S9-mix (24-hour treatment), excessive cytotoxicity was observed at 125 μg/mL and above with no surviving cells detected after the treatment. In the evaluated concentrations no statistically significant increases in the numerical value of the mutation frequency compared to the negative (vehicle) control were observed at any examined concentrations and there was no dose response to the treatment (a trend analysis showed no effect of treatment).

 

In conclusion, no mutagenic effect of the test item was observed either in the presence or absence of metabolic activation system under the conditions of this HPRT assay.

In vivo mouse micronucleus test: A study was performed in accordance with OECD 474 and EU Method B.12 to assess the potential of the test item to produce damage to chromosomes and/or aneuploidy when administered via the intraperitoneal route to mice. Following a preliminary range-finding study to confirm the toxicity of the test material and route of administration, the micronucleus study was conducted using the intrperitoneal route in groups of ten mice (five male and five female) at 50 mg/kg, 100 mg/kg and the maximum tolerated dose of 200 mg/kg. Animals were killed 24 or 48 hours later, the bone marrow was extracted and smear preparations were made and stained. Polychromatic and normochromatic erythrocytes were scored for the presence of micronuclei. Further groups of mice were given a single intraperitoneal dose of distilled water to serve as vehicle controls or dosed orally with cyclophosphamide to serve as positive controls. There was no evidence of an increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with test material when compared to the concurrent vehicle control group. No significnat change in the PCE/NCE ratio was observed after dosing with test item although the presence of clinical observations indicated that systemic absorption had occurred. The positive control material produced a marked increase in the frequency of micronucleated polychromatic erythrocytes. The test material was considered to be non-genotoxic under the conditions of the test.

Justification for classification or non-classification

The test material was found to be non-mutagenic in a bacterial reverse mutation assay, was shown to be non-clastogenic to human cells in vitro via a chromosome aberration test using human lymphocytes,non-genotoxic in an in vitro mammalian cell gene mutation test and was determined to be non-genotoxic in vivo during a mouse micronucleus test. Classification is therefore not required.