Tetrasodium 3,3'-[carbonylbis[imino(5-methoxy-2-methyl-4,1-phenylene)azo]]bis(naphthalene-1,5-disulphonate)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an Ames test that included the Prival and Mitchell (1982) modification, the substance was tested in Salmonella typhimurium strains TA1535, TA1537, TA98, TA102 and TA100 in presence and absence of metabolic activation. A strong increase in mutant frequency was reported in TA1535, TA1537, TA98 and TA100 both in presence and absence of metabolic activation. In TA102 a small but significant increase in mutant frequency was seen at 5000 ug/plate without metabolic activation and at concentrations of 500 ug/plate and above with metabolic activation. The substance is considered to be mutagenic under the conditions of this test.

The substance was tested in the L5178Y TK +/- Mouse  Lymphoma Assay according to OECD 490 up to precipitation level. Exposure was either 4 hours (with and without metabolic activation) or 24 hours (without metabolic activation). No increase in the mutant frequency at the TK +/- locus was reported (evaluation with the Global Evaluation Factor (GEF) of 126 E-06). Therefore it is concluded that the substance is non-mutagenic in this assay.

The substance was tested in an in vitro micronucleus test (OECD 487). Human lymphocytes were exposed for 4 hours (with and without metabolic activation) or 24 hours (without metabolic activation). After the exposure cytokinesis was inhibited by treatment with Cytochalasin B. There was no inhibition of the CBPI and the concentrations to be evaluated were limited by precipitate at 125 ug/mL (4 hour exposures) or 500 ug/mL (24 hours exposure). No increase in the percentage of micronucleated binuclear cells was observed in any of the treatments. Therefore it can be concluded that the substance is not clastogenic.

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:

19 October 2016 to 8 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Principles of method if other than guideline:

includes the Prival and Mitchell (1982) modification to assess the mutagenic activity of azo dyes. This part was not conducted due to clear positive results in the first test using rat S9

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

the concentrations were corrected for 7.9% water

Target gene:

histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: University of California, Berkeley, on culture discs, on 04 August 1995
- Methods for maintenance in cell culture if applicable: stored at -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34

Metabolic activation:

with and without

Metabolic activation system:

test 1 : phenobarbital/beta-naphthaflavone induced rat liver S9

Test concentrations with justification for top dose:

Test 1 (pre-incubation with rat S-9): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: substance fully soluble up to 50 mg/L

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

mitomycin C

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 30 min at 37 ± 3 °C
- Exposure duration: 48 h at 37 ± 3 °C

NUMBER OF REPLICATIONS: 3/concentration

DETERMINATION OF CYTOTOXICITY
- Method: visible reduction in the growth of the bacterial background lawn

Evaluation criteria:

a substance is considered positive when:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. 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)).

Statistics:

Dunnetts Regression Analysis

Key result

Species / strain:

other: TA1535, TA1537, TA98, TA102 and TA100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

see tables attached

An orange test item induced colouration was noted from 50 µg/plate.  No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

Large, dose-related and statistically significant increases in the frequency of TA100, TA1535, TA1537 and TA98 revertant colonies were initially observed in the absence of S9-mix from 15 µg/plate (TA1535) and in the presence of S9-mix from 150 µg/plate (TA1535). The increases observed for each bacterial strain were very large at the upper test item dose levels and very much in excess of the in-house historical untreated/vehicle control ranges for each strain with a maximum increase in excess of 100-fold over the concurrent vehicle control noted for TA1535 dosed in the absence of S9-mix.  

In TA102 a small but significant increase in mutant frequency was seen at 5000 ug/plate without metabolic activation and at concentrations of 500 ug/plate and above with metabolic activation.

All validity criteria were fulfilled.

Conclusions:

Based on the findings in this test the substance is considered mutagenic in bacteria

Executive summary:

In an Ames test the substance was tested in Salmonella typhimurium strains TA1535, TA1537, TA98, TA102 and TA100 in presence and absence of metabolic activation. A strong increase in mutant frequency was reported in TA1535, TA1537, TA98 and TA100 both in presence and absence of metabolic activation. In TA102 a small but significant increase in mutant frequency was seen at 5000 ug/plate without metabolic activation and at concentrations of 500 ug/plate and above with metabolic activation. The substance is considered to be mutagenic under the conditions of this test.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 October 2016 to 16 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

NA

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: blood of healthy non-snoking volunteers
- Cell cycle length: 16 hours
- Sex, age and number of blood donors: Preliminary Toxicity Test: female, aged 25 years; Main Experiment: female, aged 24 years
- Cell type: lymphocytes of fresh heparinized whole blood (stimulated to divide by PHA)

CULTURE MEDIA USED: Whole blood cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% fetal bovine serum (FBS), at approximately 37 ºC with 5% CO2 inhumidified air.

Culture medium lymphocytes:
8.05-9.05 mL MEM, 10% (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.75 mL heparinized whole blood

Cytokinesis block (if used):

Cytochalasin B added to block actin polymerisation

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/beta-naphthaflavone induced rat liver S9

Test concentrations with justification for top dose:

4-hour without S9 0*, 15.63*, 31.25*, 62.5*, 125*, 250, 500,
4-hour with S9 (2%) 0*, 15.63*, 31.25*, 62.5*, 125*, 250, 500,
24-hour without S9 0*, 15.63, 31.25, 62.5*, 125*, 250*, 500*

* evaluated concentration

Vehicle / solvent:

Minimal Essential Medium (MEM)

Untreated negative controls:

yes

Remarks:

MEM

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Demecolcine

Details on test system and experimental conditions:

Cells were exposed to the substance in MEM in presence and/or absence of S9 for 4 or 24 hours at approximately 37 ºC. Thereafter the cultures were centrifuged, the treatment medium was replaced with original culture medium, supplemented with Cytochalasin B at a final concentration of 4.5 µg/mL, and then incubated for a further 24 hours.Thereafter cells were fixed with methanol/glacial acetic acid (19:1 v/v) and stored at. 4 ºC prior to slide making. Slides were stained with Giemsa and dried. Thereafter cells (500 per culture) were scored for mono-, bi- and multinuclei and CPBI (plus cytostasis). In the main experiment 1000 cells per culture (2 cultures per concentration) were also scored for the number of micronucleated cells (and the number of micronuclei per cell).

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: 2000 binucleated cells was analyzed per concentration (1000 binucleated cells per culture, two cultures per concentration). Cells with 1, 2 or more micronuclei were recorded as such but the primary analysis was on the combined data. The criteria for identifying micronuclei were that they were round or oval in shape, non-refractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei. Binucleate cells were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei could be attached by a fine nucleoplasmic bridge which was approximately no greater than one quarter of the nuclear diameter.

DETERMINATION OF CYTOTOXICITY: 500 cells per culture were scored for the incidence of mononucleate, binucleate and multinucleate cells and the CBPI (Cytokinesis Block Proliferation Index) value expressed as a percentage of the vehicle controls. Cytostasis was calculated from these values

Evaluation criteria:

Negative when:
1. None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is no dose-related increase.
3. The results in all evaluated dose groups should be within the range of the laboratory historical control data.

Positive when:
1. At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is an increase which can be considered to be dose-related.
3. The results are substantially outside the range of the laboratory historical negative control data.

Statistics:

Chi-squared Test

Key result

Species / strain:

lymphocytes:

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

concentrations limited by precipitate at 125 ug/mL (4h) and 500 ug/mL (24 h)

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes:

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

concentrations limited by precipitate at 125 ug/mL

Untreated negative controls validity:

valid

Remarks:

one replicate was outside the historical control range

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: 7.23-7.33
- Effects of osmolality: 275-287 mOsm


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: available in the report
- Negative (solvent/vehicle) historical control data: available in the report

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI

Remarks on result:

other: 4 and 24 hour exposure

4-h without metabolic activation

Dose Level (µg/mL)	Mean CBPI	% Cytostasis	Mean % Binucleated cells with MN
0	1.59	0	0.65
15.63	1.65	0	0.40
62.5	1.61	0	0.55
31.25	1.63	0	0.25
125 P	1.66	0	0.65
MMC 0.2	1.46	23	5.70 ***

 

4-h with metabolic activation

Dose Level (µg/mL)	Mean CBPI	% Cytostasis	Mean % Binucleated cells with MN
0	1.53	0	1.30
15.63	1.56	0	0.95
62.5	1.51	4	1.50
31.25	1.50	6	0.90
125 P	1.53	0	0.65
CP 5	1.18	67	4.60***

 

24-h without metabolic activation

Dose Level (µg/mL)	Mean CBPI	% Cytostasis	Mean % Binucleated cells with MN
0	1.61	0	0.50
62.5	1.62	0	0.65
125	1.65	0	0.45
250	1.67	0	0.35
500	1.52	16	0.45
DC 0.075	1.46	25	2.25***

 

*** P<0.001

P= precipitate

Conclusions:

In the in vitro micronucleus assay the substance did not show clastogenic effects

Executive summary:

The substance was tested in an in vitro micronucleus test (OECD 487). Human lymphocytes were exposed for 4 hours (with and without metabolic activatin) or 24 hours (without metabolic activation). After the exposure cytokinesis was inhibited by treatment with Cytochalasin B. There was no inhibition of the CBPI and the concentrations to be evaluated were limited by precipitate at 125 ug/mL (4 hour exposures) or 500 ug/mL (24 hours exposure). No increase in the percentage of micronucleated binuclear cells was observed in any of the treatments. Therefore it can be concluded that the substance is not clastogenic.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 October 2016 to 30 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

``Kanpoan No. 287 - - Environment Protection Agency``
``Eisei No. 127 - - Ministry of Health and Welfare``
``Heisei 09/10/31 Kikyoku No. 2 - - Ministry of International Trade & Industry``

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro gene mutation study in mammalian cells

Target gene:

TK

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED: L5178Y TK+/- 3.7.2c mouse lymphoma cell line
- Source of cells: MRC Cell Mutation Unit at the University of Sussex, Brighton, UK
- Cell cycle length:ca 12 hours
- Methods for maintenance in cell culture if applicable: stored in liquid nitrogen at approximately -196°C

MEDIA USED
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes, before freezing

Additional strain / cell type characteristics:

other: TK+/-

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/beta-naphthaflavone induced rat liver S9

Test concentrations with justification for top dose:

4 h exposure: 0.24, 0.49, 0.98, 1.95, 3.91, 7.81 µg/mL
24 h exposure: 0.98, 1.95, 3.91, 7.81, 15.63, 31.25 µg/mL

Top dose based on presence of precipitate

Vehicle / solvent:

RPMI 1640 medium (R0)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

A pretest was conducted on cell cultures at 5 x 10 E5 cells/mL, using a 4-hour exposure period both with and without metabolic activation (S9), and at 1.5 x 10E5 cells/mL using a 24-hour exposure period without S9. The dose range was set at 7.81 to 2000 µg/mL. After exposure cells were washed twice, resuspended and counted with a Coulter counter. The cultures were serially diluted to 2 x 10E5 cells/mL. andincubated at 37 C with 5% CO2 in air and sub-cultured after 24 hours. After a further 24 hours the cultures were counted to assess Suspension Growth (SG)

In the main study exposures were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) in R0 or R10 medium (total volume to 20 mL). The solutions were incubated at 37°C for 4 or 24 hours with continuous shaking. the initial cell number was 1 x 10E6 cells/mL for 4 hour exposures and 0.3 x 10E6 cells/mL for 24 hour exposure.
At the end of the treatment period, for each experiment, the cells were washed twice, resuspended in R20 medium and incubated at 37°C with 5% CO2 (subcultured and counted every 24 hours -> Relative Suspension Growth (%RSG)) for 2 days.

On Day 2 of the experiment, the cells were counted again, diluted to 10E4 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL 5-trifluorothymidine (TFT) in 96-well microtitre plates. These plates were scored after incubation at 37°C with 5% CO2 for the presence of large and small colonies. On day additional cells were diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium.

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.

Statistics:

Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS (Robinson W D et al., 1989).

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no (7.23-7.33)
- Effects of osmolality: no (277-287 mOs)
- Precipitation:
main study: at 7.81 µg/mL in the 4-hour cultures and at and above 15.63 µg/mL in the 24-hour culture

RANGE-FINDING/SCREENING STUDIES: no cytotoxicity, precipitate at at 7.81 µg/mL in the 4-hour cultures and at and above 31.25 µg/mL in the 24-hour culture

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%) are reported in an annex to the report

Remarks on result:

other: 4 hour exposure

Treatment (µg/ml)	4-hours -S-9					Treatment (µg/ml)	4-hours +S-9					Treatment (µg/ml)		24-hours -S-9
		%RSG	RTG	MF§				%RSG	RTG	MF§				%RSG	RTG	MF§
0		100	1.00	151.47		0		100	1.00	163.36		0		100	1.00	126.39
0.06	Ø	93				0.06	Ø	97				0.24	Ø	93
0.12	Ø	104				0.12	Ø	108				0.49	Ø	86
0.24		102	1.07	132.42		0.24		106	1.13	122.13		0.98		96	1.06	95.71
0.49		98	0.92	143.78		0.49		96	1.27	103.98		1.95		97	1.05	99.10
0.98		101	1.19	131.16		0.98		102	1.18	122.73		3.91		88	0.95	127.40
1.95		98	1.06	155.25		1.95		96	1.29	102.95		7.81		94	1.00	140.35
3.91		100	1.09	137.43		3.91		93	0.96	142.45		15.63		85	0.90	131.22
7.81		88	0.98	126.26		7.81		95	1.05	142.60		31.25		88	0.93	121.99
	Linear	trend		NS			Linear	trend		NS			Linear	trend		NS
EMS						CP						EMS
400		67	0.47	1405.39		1.5		78	0.49	879.70		150		38	0.46	965.03

     § Positive wells per tray, 96 wells plated

Conclusions:

The substance is considered non-mutagenic in the L5178Y TK +/- Mouse Lymphoma Assay

Executive summary:

The substance was tested in the L5178Y TK +/- Mouse  Lymphoma Assay according to OECD 490 up to precipitation level. Exposure was either 4 hours (with and without metabolic activation) or 24 hours (without metabolic activation). No increase in the mutant frequency at the TK +/- locus was reported (evaluation with the Global Evaluation Factor (GEF) of 126 E-06). Therefore it is concluded that the substance is non-mutagenic in this assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

A Comet assay was performed on male rats that were dosed by gavage for 45 days in an OECD 422 study at 0, 30, 100 and 200/125 mg/kg bw. Rats that remained untreated until day 43 and were treated thereafter until day 45 with 2.5 mg/kg bw N-Nitroso-N-methylurea.

The Comet assay assessment revealed there was a small but statistically significant dose related increases in the percentage tail intensity and median percentage tail intensity of the liver but these were all within the background control ranges for the vehicle control.  The increases were considered to be due to cytotoxicity rather than DNA strand breakage, this was confirmed by the results of the histopathology on the liver where degeneration, necrosis and apoptosis were observed. Therefore the substance was considered not to induce any DNA damage in the liver.  It was concluded that there were no toxicologically significant increases in the mean or median percentage tail intensity values in the jejunum, glandular stomach and liver and therefore the substance was considered to be non-genotoxic to these tissues.  

Link to relevant study records

Reference

Endpoint:

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

Remarks:

Comet Assay

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This in vivo study was performed as part of the OECD 422 study. This has been done as based on the positive outcome of the Ames test, an additional in vivo study is warranted. In order to minimize the number of animals necessary for the assessment and to further investigate the mutagenicity endpoint the assessment was incorporated in the repeated dose study as is also described in the guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Principles of method if other than guideline:

Six males from each dose group will be dosed to Day 45 and six N-Nitroso-N-methylurea dosed controls will kept without dosing during day 0-43 and will be dosed from day 43 to 45. For the males from Groups 1 to 4 of the OECD 422 study as well as all positive control males, samples of the liver (after recording of liver weight), glandular stomach and jejunum were processed to provide single cell suspensions with sufficient numbers of cells for the Comet Assay. The procedure was performed under subdued lighting and the Comet assay tissues/cells were processed as quickly as possible, using ice-cold buffers to maintain the tissues and cell preparations at low temperature.

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:

TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK
- Strain: Wistar Han™:RccHan™:WIST
- Age at study initiation: 11 weeks
- Weight at study initiation: 274-361 g
- Housing: group housed up to three in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK) (except during the mating period of the OECD 422 study)
- Diet: pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.) ad libitum
- Water: ad libitum
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): at lest 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil for treatment groups (4 mL/kg), distilled water for positive control (N-Nitroso-N-methylurea ,10 mL/kg)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
the test item was prepared at the appropriate concentrations as a suspension in Corn oil. Formulations were made daily from Days 1 to 8 of dosing, and dosed within four hours of being prepared. Following confirmed stability and homogeneity results, formulations were prepared weekly and stored refrigerated in the dark.
N-Nitroso-N-methylurea was dissolved in distilled water at appropriate concentration. A purity adjustment (for 90% purity) was made when preparing dosing formulations. Fresh formulations were prepared each day and dosed within two hours of preparation.

Duration of treatment / exposure:

45 days for treatment groups, 3 days for positive control (undosed during the first 43 days)

Frequency of treatment:

daily

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

125 mg/kg bw/day (nominal)

Remarks:

initially dosed at 200 mg/kg bw during day 1-22, reduced to 125 mg/kg bw thereafter (due to mortality and sever weight loss)

Dose / conc.:

2.5 mg/kg bw/day (actual dose received)

Remarks:

N-Nitroso-N-methylurea (positive control)

No. of animals per sex per dose:

6 males for treatment groups, 5 males for positive controls

Control animals:

yes, concurrent vehicle

yes, historical

Positive control(s):

N-Nitroso-N-methylurea
- Justification for choice of positive control(s): according to the guideline and historical control data are available in the laboratory
- Route of administration: gavage
- Doses / concentrations: 2.5 mg/kg bw

Tissues and cell types examined:

glandular stomach, jejunum and liver

Details of tissue and slide preparation:

Liver - A small piece of liver was excised (approximately 1 cm2) 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 and Jejunum - 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 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.
A section of jejunum (approximately 2 cm2) was removed, cleaned and then immersed in stomach buffer for approximately 15 minutes on ice. A cell suspension was obtained by scraping the tissue of the jejunum into stomach buffer and filtering it through gauze.

Adequate numbers of slides were pre-coated with 0.5% normal melting point agarose and stored at room temperature. The slides were labelled for animal number, study number and tissue type prior to use for the Comet assay.
Once the cell suspensions were obtained, approximately 30 μL of the cell suspension 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. The agarose/cell suspension mix was immediately covered with a glass coverslip, transferred to a cold room at approximately 4 °C in the dark for approximately 20 minutes to allow it to solidify.
Once 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 overnight. All slides went through the subsequent processing.
After the lysis phase had been completed the slides were removed from the lysing buffer, rinsed with neutralization buffer (0.4M Tris pH 7.5) to remove residual detergents and salts and then placed randomly into an electrophoresis unit. The electrophoresis unit was filled with chilled electrophoresis buffer (pH >13) until the slide surface was just covered. The slides were left for approximately 20 minutes to allow the DNA to unwind, after which electrophoresis proceeded at approximately 0.7 V/cm (calculated between the electrodes), 300 mA for 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 to ensure the electrophoresis solution was maintained at low temperature (2-10 °C).
At the end of the electrophoresis period the bath was switched off, the slides gently removed and rinsed three times with neutralization buffer for approximately 5 minutes each time. The slides were then carefully drained and fixed in cold 100% methanol for 5 minutes and allowed to air dry, after which they were stored prior to staining and scoring. For each tissue, two processed slide gels were scored blindly. To each dry slide gel, 75 μL of propidium iodide (20 μg/mL) was placed on top of the slide and overlaid with a clean cover slip. After a short period to allow hydration and staining of the DNA, the slide was placed onto the stage of a fluorescence microscope and scored for comets using a CCD camera attached to a PCbased image analysis program (Comet IV version 4.3.1). Two slide gels for each tissue for each animal were scored with a maximum of 100 cells per slide gel giving an accumulative total of 200 cells per tissue per ani.

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if:
a) at least one of the test doses exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is considered to be dose-related
c) the results are substantially outside the distribution of the historical negative control range
The test chemical is when all three conditions are met considered to be able to induce DNA strand breakage in the tissues studied

Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no evidene of a dose-related response
c) all results are within historical negative control range
d) direct or indirect evidence to demonstrate exposure of, or toxicity to, the target tissue(s) has been achieved
The test chemical is then considered unable to induce DNA strand breakage in the tissues studied in this test system.

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

liver degeneration, necrosis and apoptosis, jejunum yellow discolouration; stomach green/yellow coloured contents

Vehicle controls validity:

valid

Remarks:

within historical control ranges

Positive controls validity:

valid

Remarks:

within or slightly above historical control values

Additional information on results:

At 100 or 200/125 mg/kg bw/day there were small but statistically significant dose related increases (p<0.05 - p<0.01) in the mean and median percentage tail intensities over the vehicle control value for the liver. However, these increases were all within the laboratory historical control range for the vehicle used, and were set against a relatively low vehicle control value and were therefore considered to be due to cytotoxicity rather than DNA strand breakage. This is confirmed by the results of the histopathology on the liver where degeneration, necrosis and apoptosis were observed. The treatment with the test item was therefore considered not to induce any DNA damage in the liver.

Glandular Stomach

Dose level (mg/kg bw)	Group mean % hedgehogs	Group mean % tail intensity	Group mean of median % tail intensity per animal	Group mean SD % tail intensity
0	4.27	2.35	0.58	4.18
30	4.74	2.08	0.49	3.66
100	5.83	2.72	0.82	4.21
200/125	4.42	2.25	0.63	3.70
25 (MNU)	9.61	34.01	32.32	13.71

 

Jejunum

Dose level (mg/kg bw)	Group mean % hedgehogs	Group mean % tail intensity	Group mean of median % tail intensity per animal	Group mean SD % tail intensity
0	8.72	3.48	1.07	6.06
30	11.05	4.03	1.48	6.06
100	9.25	3.63	1.18	6.01
200/125	10.74	4.00	1.32	6.79
25 (MNU)	16.59	47.53	48.22	15.44

 

Liver

Dose level (mg/kg bw)	Group mean % hedgehogs	Group mean % tail intensity	Group mean of median % tail intensity per animal	Group mean SD % tail intensity
0	0	0.35	0.02	0.85
30	0	0.40	0.03	0.97
100	0	0.52*	0.03*	1.45
200/125	0	0.54**	0.04**	1.27
25 (MNU)	15.33	43.05	43.07	9.09

*P<0.05; ** p<0.01

Historical controls Liver (based on 15-16 experiments)

Dose level (mg/kg bw)	Group mean % hedgehogs	Group mean % tail intensity	Group mean of median % tail intensity per animal	SD % of tail intensity
vehicle	0.00-1.16	0.17-0.95	0.00-0.08	0.55-5.86
Positive control	0.29-10.24	13.65-46.47	13.49-47.10	5.15-13.31

 

Conclusions:

The substance is considered not to induce toxicologically significant increases in the mean or median percentage tail intensity values in the jejunum, glandular stomach and liver and therefore the substance was considered to be non-genotoxic to these tissues.  

Executive summary:

A Comet assay was performed on male rats that were dosed by gavage for 45 days in an OECD 422 study at 0, 30, 100 and 200/125 mg/kg bw. Rats that remained untreated until day 43 and were treated thereafter until day 45 with 2.5 mg/kg bw N-Nitroso-N-methylurea.

The Comet assay assessment revealed there was a small but statistically significant dose related increases in the percentage tail intensity and median percentage tail intensity of the liver but these were all within the background control ranges for the vehicle control.  The increases were considered to be due to cytotoxicity rather than DNA strand breakage. This was confirmed by the results of the histopathology on the liver where degeneration, necrosis and apoptosis were observed. Therefore the substance was considered not to induce any DNA damage in the liver.  It was concluded that there were no toxicologically significant increases in the mean or median percentage tail intensity values in the jejunum, glandular stomach and liver and therefore the substance was considered to be non-genotoxic to these tissues.  

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Based on the outcome of the Ames test an in vivo Comet assay is the standard next step in the assessment of potential mutagenic effects of the substance. Therefore the in vivo Comet assay was included in the study design for the OECD 422 study in order to avoid unnecessary additional vertebrate testing.

Justification for classification or non-classification

Based on the information as found in the in vitro tests and the in vivo Comet assay as tested as part of the OECD 422 study, it can be concluded that the substance is not mutagenic and that no classification according to Regulation (EC) No 1272/2008 is needed.