Trisodium [3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulphonato(3-)][2-hydroxy-3-[(2-hydroxy-1-naphthyl)azo]-5-nitrobenzenesulphonato(3-)]chromate(3-)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Read-Across - OECD TG 471 - supporting study - KS2 - with and without S9: positive.

Read-Across - OECD TG 471 - supporting study - KS2 - with and without S9: positive.

Read-Across - OECD TG 471 NR deficient strains - key study - KS1 - with and without S9: positive.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

other: read-across on supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2020 - 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

nitroreductase-deficient strains TA98NR and TA100NR

Principles of method if other than guideline:

The objective of this study was to evaluate the ability of test item to induce reverse mutations in histidine-requiring strains of Salmonella typhimurium in the absence and presence of a reductive hamster liver metabolising system (S-9). By assessing the mutagenicity of test item in nitroreductase deficient strains (TA98NR and TA100NR) alongside parent nitroreductase competent strains (TA98 and TA100), the role of nitroreduction in any test article related mutagenic activity could be determined.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Additional strain / cell type characteristics:

nitroreductase deficient

Remarks:

TA98NR and TA100NR

Metabolic activation:

with and without

Metabolic activation system:

The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was obtained from Molecular Toxicology Incorporated, USA where it was prepared from uninduced male Golden Syrian hamsters. The S-9 was stored frozen at <-50°C, and thawed prior to use. Each batch was checked by the manufacturer for sterility, protein content, ability to convert ethidium bromide and cyclophosphamide to bacterial mutagens, and cytochrome P 450-catalysed enzyme activities (alkoxyresorufin-O-dealkylase activities

Test concentrations with justification for top dose:

5, 16, 50, 160, 500, 1600 and 5000 µg/plate

A maximum concentration of 5000 µg/plate was selected for Mutation Experiment treatmenty, in order that treatments were performed up to this maximum recommended concentration according to current regulatory guidelines OECD 471.

Vehicle / solvent:

water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

congo red

mitomycin C

other: Metronidazole, 2-aminoanthracene

Details on test system and experimental conditions:

pre-incubation methodology in the absence and presence of a modified (reductive) S-9 mix. These platings were achieved by the following sequence of additions to sterile pre incubation tubes:
• 0.1 mL of bacterial culture
• 0.1 mL of test article solution/vehicle control or 0.05 mL of positive control
• 0.5 mL of 30% reductive S-9 mix or buffer solution
Quantities of test article or control solution, bacteria and S-9 mix or buffer solution detailed above, were mixed together and placed in an orbital incubator set to either 37°C (for the treatments in the absence of S-9) or 30°C (for treatments in the presence of S 9) for 30 minutes, before the addition of 2 mL of supplemented molten agar at 45±1°C followed by rapid mixing and pouring on to Vogel-Bonner E agar plates.
When set, the plates were inverted and incubated protected from light for 3 days in an incubator set to 37°C. Following incubation, these plates were examined for evidence of cytotoxicity to the background lawn, and where possible revertant colonies were counted.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic if:
1. A concentration related increase in revertant numbers was ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98, TA98NR, TA100 or TA100NR) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values
2. The positive trend/effects described above were reproducible.
The test article was considered positive in this assay if both of the above criteria were met.
The test article was considered negative in this assay if neither of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Biological relevance was taken into account, for example consistency of response within and between concentrations and between experiments

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

bacteria, other: TA 98NR

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

bacteria, other: TA 98NR

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

bacteria, other: TA100NR

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The objective of this study was to evaluate the ability of test item to induce reverse mutations in histidine-requiring strains of Salmonella typhimurium in the absence and presence of a reductive hamster liver metabolising system (S-9). By assessing the mutagenicity of the test substance in nitroreductase deficient strains (TA98NR and TA100NR) alongside parent nitroreductase competent strains (TA98 and TA100), the role of nitroreduction in any test article related mutagenic activity could be determined.
The results shows that in the absence of S-9, as a clear mutagenic response was observed in strain TA98 but not in strain TA98NR, nitroreduction appears to be playing a key role in the mutagenicity of test item.

Mutation Experiment 1 treatments of all the tester strains were performed using a pre incubation methodology in the absence and presence of a modified (reductive) S 9 mix using final concentrations of test item at 5, 16, 50, 160, 500, 1600 and 5000 µg/plate, plus vehicle and positive controls. Following these treatments, no clear evidence of cytotoxicity was observed, as would usually be manifest by a diminution of the background bacterial lawn and/or a marked reduction in revertant numbers. Reductions in what appeared to be mutagenic responses at the higher concentrations in some strains were however considered likely to have been due to test article-related cytotoxicity.

 

Following test item treatments of all the tester strains, notable increases in revertant numbers were observed in strains TA98, TA100 and TA1537 in the absence of S-9 and in strains TA98NR in the presence of S-9 only. These increase all approached or exceeded 2-fold the concurrent vehicle control level, and provided at least some evidence of a concentration-relationship, although in almost all cases the maximum increase was observed below the maximum treatment concentration, but it was considered that this was likely due to test article-related cytotoxicity. By far the largest magnitude of increases occurred in strain TA98NR in the presence of S-9, and but further experimentation is required to understand the reason behind this finding.

 

Clear mutagenic responses in the absence of S-9 were observed in strain TA98, TA100 and TA537 with no clear responses observed following comparable treatments with the corresponding nitroreductase deficient strain TA98NR and TA100NR, this is considered indicative that nitroreduction is involved in the observed mutagenicity in the absence of S-9. However, in the presence of S-9 a much larger magnitude of increase was observed in strain TA98NR compared to comparable treatments in strain TA98. It may be that for treatments performed in the presence of S-9, the enzymes in the S-9 are affecting the impact that nitroreduction has on the mutagenic activity. It is therefore considered that the involvement of nitroreduction in the mutagenicity of the test substance in the presence of S-9 is unclear, and further experimentation is required to elucidate the mechanism of mutagenicity under these treatment conditions.

 

Finally, all the data can be considered Acceptable and Valide According to the criteria mentioned above.

Conclusions:

It was concluded that the tested substance induced mutation in histidine-requiring Salmonella typhimurium strain TA98NR in the presence of a reductive hamster liver metabolic activation system (S-9), and in strains TA98, TA100 and TA1537 in the absence of S-9, when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 μg/plate (the maximum recommended concentration according to current regulatory guidelines). The relative reduced mutagenic responses in the absence of S-9 with the nitroreductase deficient strains compared to those with the parent strains indicate that nitroreduction plays a significant role in the mutagenic activity of the tested substance, but the mechanism of mutagenicity in the presence of S-9 remains unclear.

Executive summary:

The substance was assayed for mutation in seven histidine-requiring strains (TA98, TA100, TA1535, TA1537, TA102, TA98NR and TA100NR) of Salmonella typhimurium, both in the absence and in the presence of a reductive hamster liver metabolising system (S-9) in a single experiment. All treatments in this study were performed using formulations prepared in purified water. As the test substance is an azo compound, testing in the presence of S-9 in this study was performed using a modified reductive (Prival) S-9 pre-incubation methodology, as it is known that azo compounds can be reduced to free aromatic amines, which can be mutagenic.
Mutation Experiment treatments of all the tester strains were performed using a pre-incubation methodology in the absence and presence of a modified (reductive) S-9 mix using final concentrations of the test substance at 5, 16, 50, 160, 500, 1600 and 5000 μg/plate. Following these treatments, clear evidence of cytotoxicity was observed at 5000 μg/plate in strain TA102 in the absence of S-9 only.
It should be noted that due to uncharacteristic vehicle control data following the initial Mutation Experiment treatments of strain TA98NR in the absence and presence of S-9, these treatments were repeated to provide valid mutation data for this strain. In order that concurrent mutation data were available for direct comparison of any mutagenic response in this strain with the parent strain, strain TA98 treatments were also repeated alongside the strain TA98NR repeat treatments. As mutation data from the initial strain TA98 treatments in the absence and presence of S-9 were valid, data from these further TA98 treatments are reported as further Mutation Experiment data.
The test article was completely soluble in the aqueous assay system at all concentrations treated.
Vehicle and positive control treatments were included for all strains. The mean numbers of revertant colonies were comparable with acceptable ranges for vehicle control treatments, and the responses with the positive control treatments were sufficient to confirm the correct strain and assay functioning.
Following treatments of all the test strains with the test substance, notable increases in revertant numbers were observed in strain TA98 in the absence and presence of S-9, in strains TA100 and TA1537 in the absence of S-9 only and in strain TA98NR in the presence of S-9 only. In strains TA100 and TA1537 in the absence of S-9 and in strain TA98NR in the presence of S-9, concentration-related increases that exceeded 2-fold (strains TA100, TA98 and TA98NR) or 3-fold (strain TA1537) the vehicle control level were observed, although in some cases the responses ‘tailed off’ at the highest concentration(s), which was considered likely to have been due to test article related cytotoxicity. All these increases were sufficient to be considered as clear evidence of test substance mutagenic activity in these strains. In strain TA98 in the presence of S-9, concentration-related increases that ‘tailed off’ at the highest concentration(s) were observed, but the increase only exceeded 2-fold the concurrent vehicle control level with the further Mutation Experiment treatments. In strain TA98NR in the absence of S-9, an increase in revertant numbers was observed that provided at least some evidence of a concentration-relationship. This increase fell slightly below the 2-fold threshold for an increase to be considered as clear evidence of mutagenic activity in this strain, and therefore along with the increases seen in strain TA98 in the presence of S-9 may have been further evidence of the mutagenic activity described previously. In strain TA102 in the presence of S-9, a 1.4-fold increase was seen. This increase was only evident at a single intermediate treatment concentration, and was therefore not considered to be a clearly concentration-related effect. Together with the relatively small magnitude of the increase (below 1.5-fold the concurrent vehicle control level), this increase was not sufficient to be considered as clear evidence of mutagenic activity.
When the relative magnitude of the responses in the absence of S-9 between the nitroreductase deficient strains and their respective parent strains were assessed, there was a markedly greater increase in strain TA100 than in TA100NR, as no notable increase was observed with the nitroreductase deficient strain. The increases in strain TA98 (in both the initial and further treatments) were slightly greater in magnitude, and occurred at a lower treatment concentration, than the increase observed in strain TA98NR. As there is still some low level residual nitroreductase activity present in strains TA98NR and TA100NR (Rosenkranz and Mermelstein (1983)), complete elimination of any mutagenic response was not expected in these strains, although this did appear to occur with strain TA100NR in the absence of S-9. These data were considered to indicate that nitroreduction played at least some part in the mutagenic activity observed in the absence of S-9.
In the presence of S-9, no notable increases were observed in strains TA100 or TA100NR, so only the relative responses in strains TA98 and TA98NR provided an indication of the role that nitroeduction may play in the observed mutagenic activity. A clear mutagenic response was only observed in strain TA98NR, with only small increases seen in strain TA98. It is unclear why an increased response should occur in the absence of nitroreduction, but it is known that the enzymes present in S-9 can result in confounding effects when trying to assess the role of an individual enzyme (such as nitroreductase) in the mutagenic activity of a compound. It is therefore considered that the involvement of nitroreduction in the mutagenicity of the test substance in the presence of S-9 is unclear, and further experimentation is required to elucidate the mechanism of mutagenicity under these treatment conditions. It was concluded that the test substance induced mutation in histidine-requiring Salmonella typhimurium strain TA98NR in the presence of a reductive hamster liver metabolic activation system (S-9), and in strains TA98, TA100 and TA1537 in the absence of S-9, when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 μg/plate (the maximum recommended concentration according to current regulatory guidelines). The relative reduced mutagenic responses in the absence of S-9 with the nitroreductase deficient strains compared to those with the parent strains indicate that nitroreduction plays a significant role in the mutagenic activity of the test substance, but the mechanism of mutagenicity in the presence of S-9 remains unclear.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

December, 1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Study conducted according to internationally accepted testing guidelines and performed according to the GLP. The OECD recommended combination of strains was not respected: none of the E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102 was tested. Justification for Read Across is detailed in the endpoint summary.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

GROWING AND CONSERVATION OF BACTERIAL TEST STRAINS
The bacterial strains were kept as frozen broth cultures, in aliquots of 0.5 ml at -70 °C with 8.0 % dimethylsulfoxide (DMSO).
Fresh cultures were prepared by adding 0.5 ml of a thawed stock culture to 25 ml of nutrient broth (normal Difco nutrient broth for strains TA 1535 and TA 100, and double strength Difco nutrient broth for TA 1537 and TA 98, in 0.5 % NaCI).
A nutrient agar (0.8 % Difco nutrient broth, 1.5 % Difco agar, 0.5 % NaCI) was also streaked.
The broth was incubated in the dark in a shaking water bath at 37 °C for 16 hours, while the plate was incubated at 37 °C overnight. Plates were then transferred to the refrigerator, for up to one week.

CHECKING OUT TESTER STRAINS
All strains were tested for the presence of their mutations.
a)
_The mutation in the histidine operon, basic to the test system, was tested by checking for growth in the presence and absence of histidine on a minimal medium-agar base.
_Bacteria of the nutrient agar plate were streaked on a minimal medium plate supplemented with 0.1 ml of 0.1 M L-histidine and 0.1 ml of 0.5 mM biotin as a growth requirement.
_The plates were incubated at 37 °C overnight. Growth, for all strains, was seen only where histidine and biotin were present.
b)
Sensitivity to crystal violet is a check for the presence of the deep rough ( rfa) mutation, the loss of the lipopolysaccharide coat on the bacterial surface.
Three drops of the broth culture were spread on the surface of a nutrient agar plate. A sterile filter paper disc containing crystal violet (10 µl of a 1 mg/ml solution) was placed on this surface. A zone of inhibition around the disc, after 24 hours incubation, shows the presence of the (rfa) mutation.
c)
Two of the tested strains (TA 98 and TA 100) contain a plasmid expressing resistance to ampicillin (R factor). To check for the presence of the plasmid, a sterile filter paper disc containing ampicillin (10 µl of 8 mg/ml in 0.02 N NaOH) was placed on a nutrient agar plate spread with the broth.
In strains TA 1535 and TA 1537 a zone of inhibition occurs around the disc, but for TA 98 and TA 100 where the R factor is present, no zone of inhibition is seen.
d)
The uvrB deletion, the loss of the excision repair system, makes the bacteria sensitive to UV irradiation.
One drop of the broth was cross-streaked in a nutrient agar plate. One half of the plate was irradiated for 20 seconds under a 15 watt UV lamp at a distance of approximately 30 cm. After 24 hours' incubation, growth was found only on the unirradiated part of the streak for all strains.

INDUCTION OF RAT LIVER ENZYMES
Three male Sprague-Dawley rats weighing approximately 200 grams were given an i.p. injection of Arocior 1254 (IVIonsanto) in peanut oil at a dose of 500 mg/kg.
Five days after the injection, the rats were anaesthetized with ether, decapitated and the livers removed in a sterile manner. The livers were homogenized in three times their volume of sterile 0.15 M KCl, pooled together and centrifuged for 15 min at 9000 xg (Beekman refrigerated ultracentrifuge). The supernatant (termed the S-9 fraction) was aliquoted into sterile tubes and frozen at -70 °C.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver enzymes

Test concentrations with justification for top dose:

20, 80, 320, 1280 and 5120 µg/plate

Vehicle / solvent:

dimethylsulfoxide (DMSO)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

other: 2-anthramine (2-AA), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and daunomycidine (DM)

Remarks:

Without S9: TA 1535 and TA 100 MNNG (1.6 µg); TA 1537 9-AA(50 µg); TA 98 DM (5 µg). With S9: all strains 2-AA (12.5 µg)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

PREPARATION OF MATERIALS
The minimal-glucose agar medium base was prepared from a 1.5 % Bacto-Difco agar in Vogel & Bonner E medium with 2 % glucose.
The top agar is a 0.6 % Bacto-Difco agar, 0.5 % NaCI solution. Before use, the agar was melted in a boiling water bath and then left to equilibrate to 45 °C.
Ten ml of sterile 0.5 mM L-histidine - 0.5 mM biotin were added per 100 ml of top agar.
The compounds to be tested were prepared fresh daily in DMSO or in sterile water.
The appropriate dilutions were made from a 51.2 mg/stock.
The S-9 mix was prepared fresh daily from sterile stocks. 0.5 ml contains:
4.0 µ moles MgCl2
16.5 µ moles KCl
2.5 µ moles G-6-P
2.0 µ moles NADP
50.0 µ moles Phosphate Buffer, pH 7.4
150.0 µl liver homogenate.

The test system was as follows; the under mentioned compounds were added to sterile glass tubes in the given order:
- 0.1 ml of solution of compound to be tested
- 0.5 ml of S-9 mix (or 0.85 % saline)
- 0.1 ml of bacterial suspension (2-3 x 10^8 bacteria)
- 2 ml of molten top agar at 45 °C.
The tubes were agitated using a vortex mixer and poured onto the minimal medium base. The plates were incubated for 48 hours at 37 °C in the dark. The colonies, the revertants to the wild type, were counted manually or electronically using a Fisher Colony Counter. Each strain has a characteristic spontaneous reversion rate.
An examination under the microscope was used to verify the presence of the background produced by growth of auxotrophic bacteria on traces of histidine and biotin, or the absence of background due to a toxic effect of the test substance.

NUMBER OF REPLICATIONS
Every concentration was tested in triplicate.

Evaluation criteria:

The criteria of mutagenicity used are a doubling of the spontaneous reversion rate and a dose-effect relationship.

Species / strain:

other: S. typhimurium TA 1535, TA 1537 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

a toxic effect was observed with the strains TA 1537 at 5120 µg of product in presence and absence of S-9 mix and with the strain TA 100 at 5120 µg only in absence of S-9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

a toxic effect was noted at 5120 µg with S-9 mix and at 1280 µg and 5120 µg without S-9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Additional information on results:

Under the experimental conditions defined in the protocol, and employing a doubling of the spontaneous reversion rate and dose-effect relationship as criteria of mutagenicity, the product was mutagenic for Salmonella typhimurium strain TA 98. No mutagenic effect was noted with the strains TA 1535, TA 1537 and TA 100.

With strain TA 98 the evidence for mutagenicity existed both in the absence and presence of a liver microsomal enzyme preparation (S-9 mix) from male rats pretreated with Aroclor 1254 at 1280 pg with S-9 mix, at 80 and 320 µg without S-9 mix. A toxic effect was noted at 5120 µg with S-9 mix and at 1280 µg and 5120 µg without S-9 mix.

A toxic effect was also observed with the strains TA 1537 at 5120 µg of product in presence and absence of S-9 mix and with the strain TA 100 at 5120 µg only in absence of S-9 mix.

	With S9
	TA 98	TA 100	TA 1535	TA 1537
Spontaneous reversion	49 (SD = 2)	142 (SD = 13)	17 (SD = 3)	9 (SD = 2)
Control DMSO	34 (SD = 5)	121 (SD = 5)	14 (SD = 5)	12 (SD = 2)
Positive control	2497 (SD = 437)	2613 (SD = 78)	817 (SD = 52)	275 (SD = 26)
Test substance (µg)
20.0	57 (SD = 7)	117 (SD = 11)	13 (SD = 3)	11 (SD = 5)
80.0	59 (SD = 7)	126 (SD = 11)	12 (SD = 3)	9 (SD = 1)
320	77 (SD = 7)	135 (SD = 4)	10 (SD = 2)	10 (SD = 6)
1280.0	118 (SD = 22)	115 (SD = 9)	12 (SD = 1)	16 (SD = 4)
5120.0	<0>	174 (SD = 1)	28 (SD = 3)	<0>
	Without S9
	TA 98	TA 100	TA 1535	TA 1537
Spontaneous reversion	21 (SD = 3)	121 (SD = 10)	24 (SD = 2)	6 (SD = 2)
Control DMSO	21 (SD = 5)	100 (SD = 1)	18 (SD = 6)	4 (SD = 1)
Positive control	863 (SD = 106)	2088 (SD = 62)	2190 (SD = 46)	233 (SD = 31)
Test substance (µg)
20.0	37 (SD = 1)	93 (SD = 5)	19 (SD = 1)	5 (SD = 1)
80.0	57 (SD = 12)	123 (SD = 8)	24 (SD = 5)	8 (SD = 2)
320	97 (SD = 10)	136 (SD = 12)	31 (SD = 2)	11 (SD = 2)
1280.0	0 (SD = 0)	151 (SD = 8)	29 (SD = 4)	7 (SD = 2)
5120.0	<0>	<0>	12 (SD = 2)	<0>

Conclusions:

Interpretation of results:
positive.

A mutagenic effect was observed with the strain TA 98 (with and without metabolic activation).

Executive summary:

The substance was tested with the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 at concentrations from 20 to 5120 µg per Petri dish both in the presence and absence of metabolic activation.

Under the experimental conditions defined in the protocol, and employing a doubling of the spontaneous reversion rate and dose-effect relationship as criteria of mutagenicity, the product was mutagenic for Salmonella typhimurium strain TA 98. No mutagenic effect was noted with the strains TA 1535, TA 1537 and TA 100.

With strain TA 98 the evidence for mutagenicity existed both in the absence and presence of a liver microsomal enzyme preparation (S-9 mix) from male rats pretreated at 1280 µg with S-9 mix, at 80 and 320 µg without S-9 mix. A toxic effect was noted at 5120 µg with S-9 mix and at 1280 µg and 5120 µg without S-9 mix.

A toxic effect was also observed with the strains TA 1537 at 5120 µg of product in presence and absence of S-9 mix and with the strain TA 100 at 5120 µg only in absence of S-9 mix.

 

Conclusion:

From the results of Ames test, it appears that the test substance presents only a very slight mutagenic effect in this system. This effect is observed only with one tester strain (TA 98). This effect is markedly diminished when liver microsomial enzymes are added to the preparation suggesting that the compound might be detoxified in vivo.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

January, 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Remarks:

Study conducted according to internationally accepted testing guidelines and performed according to the GLP. The OECD recommended combination of strains was not respected: none of the E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102 was tested. Justification for Read Across is detailed in the endpoint summary.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

GROWING AND CONSERVATION OF BACTERIAL TEST STRAINS
The bacterial strains were kept as frozen broth cultures, in aliquots of 0.5 ml at -70 °C with 8.0 % dimethylsulfoxide (DMSO).
Fresh cultures were prepared by adding 0.5 ml of a thawed stock culture to 25 ml of nutrient broth (normal Difco nutrient broth for strains TA 1535 and TA 100, and double strength Difco nutrient broth for TA 1537 and TA 98, in 0.5 % NaCI).
A nutrient agar (0.8 % Difco nutrient broth, 1.5 % Difco agar, 0.5 % NaCI) was also streaked.
The broth was incubated in the dark in a shaking water bath at 37 °C for 16 hours, while the plate was incubated at 37 °C overnight. Plates were then transferred to the refrigerator, for up to one week.

CHECKING OUT TESTER STRAINS
All strains were tested for the presence of their mutations.
a)
_The mutation in the histidine operon, basic to the test system, was tested by checking for growth in the presence and absence of histidine on a minimal medium-agar base.
_Bacteria of the nutrient agar plate were streaked on a minimal medium plate supplemented with 0.1 ml of 0.1 M L-histidine and 0.1 ml of 0.5 mM biotin as a growth requirement.
_The plates were incubated at 37 °C overnight. Growth, for all strains, was seen only where histidine and biotin were present.
b)
Sensitivity to crystal violet is a check for the presence of the deep rough ( rfa) mutation, the loss of the lipopolysaccharide coat on the bacterial surface.
Three drops of the broth culture were spread on the surface of a nutrient agar plate. A sterile filter paper disc containing crystal violet (10 µl of a 1 mg/ml solution) was placed on this surface. A zone of inhibition around the disc, after 24 hours incubation, shows the presence of the (rfa) mutation.
c)
Two of the tested strains (TA 98 and TA 100) contain a plasmid expressing resistance to ampicillin (R factor). To check for the presence of the plasmid, a sterile filter paper disc containing ampicillin (10 µl of 8 mg/ml in 0.02 N NaOH) was placed on a nutrient agar plate spread with the broth.
In strains TA 1535 and TA 1537 a zone of inhibition occurs around the disc, but for TA 98 and TA 100 where the R factor is present, no zone of inhibition is seen.
d)
The uvrB deletion, the loss of the excision repair system, makes the bacteria sensitive to UV irradiation.
One drop of the broth was cross-streaked in a nutrient agar plate. One half of the plate was irradiated for 20 seconds under a 15 watt UV lamp at a distance of approximately 30 cm. After 24 hours' incubation, growth was found only on the unirradiated part of the streak for all strains.

INDUCTION OF RAT LIVER ENZYMES
Three male Sprague-Dawley rats weighing approximately 200 grams were given an i.p. injection of Arocior 1254 (IVIonsanto) in peanut oil at a dose of 500 mg/kg.
Five days after the injection, the rats were anaesthetized with ether, decapitated and the livers removed in a sterile manner. The livers were homogenized in three times their volume of sterile 0.15 M KCl, pooled together and centrifuged for 15 min at 9000 xg (Beekman refrigerated ultracentrifuge). The supernatant (termed the S-9 fraction) was aliquoted into sterile tubes and frozen at -70 °C.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver enzymes

Test concentrations with justification for top dose:

20, 80, 320, 1280 and 5120 µg/plate

Vehicle / solvent:

dimethylsulfoxide (DMSO)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

other: 2-anthramine (2-AA), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and daunomycidine (DM)

Remarks:

Without S9: TA 1535 and TA 100 MNNG (1.6 µg); TA 1537 9-AA(50 µg); TA 98 DM (5 µg). With S9: all strains 2-AA (12.5 µg)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

PREPARATION OF MATERIALS
The minimal-glucose agar medium base was prepared from a 1.5 % Bacto-Difco agar in Vogel & Bonner E medium with 2 % glucose.
The top agar is a 0.6 % Bacto-Difco agar, 0.5 % NaCI solution. Before use, the agar was melted in a boiling water bath and then left to equilibrate to 45 °C.
Ten ml of sterile 0.5 mM L-histidine - 0.5 mM biotin were added per 100 ml of top agar.
The compounds to be tested were prepared fresh daily in DMSO or in sterile water.
The appropriate dilutions were made from a 51.2 mg/stock.
The S-9 mix was prepared fresh daily from sterile stocks. 0.5 ml contains:
4.0 µ moles MgCl2
16.5 µ moles KCl
2.5 µ moles G-6-P
2.0 µ moles NADP
50.0 µ moles Phosphate Buffer, pH 7.4
150.0 µl liver homogenate.

The test system was as follows; the under mentioned compounds were added to sterile glass tubes in the given order:
- 0.1 ml of solution of compound to be tested
- 0.5 ml of S-9 mix (or 0.85 % saline)
- 0.1 ml of bacterial suspension (2-3 x 10^8 bacteria)
- 2 ml of molten top agar at 45 °C.
The tubes were agitated using a vortex mixer and poured onto the minimal medium base. The plates were incubated for 48 hours at 37 °C in the dark. The colonies, the revertants to the wild type, were counted manually or electronically using a Fisher Colony Counter. Each strain has a characteristic spontaneous reversion rate.
An examination under the microscope was used to verify the presence of the background produced by growth of auxotrophic bacteria on traces of histidine and biotin, or the absence of background due to a toxic effect of the test substance.

NUMBER OF REPLICATIONS
Every concentration was tested in triplicate.

Evaluation criteria:

The criteria of mutagenicity used are a doubling of the spontaneous reversion rate and a dose-effect relationship.

Species / strain:

other: S. typhimurium TA 1535, TA 1537 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

a toxic effect was also observed with the strains TA 1537 at 5120 µg of product in presence and absence of S-9 mix and with the strain TA 100 at 5120 µg only in absence of S-9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

a toxic effect was noted at 5120 µg with S-9 mix and at 1280 µg and 5120 µg without S-9 mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Additional information on results:

Under the experimental conditions defined in the protocol, and employing a doubling of the spontaneous reversion rate and dose-effect relationship as criteria of mutagenicity, the product was mutagenic for Salmonella typhimurium strain TA 98. No mutagenic effect was noted with the strains TA 1535, TA 1537 and TA 100.

With the strain TA 98 the evidence for mutagenicity existed only in the absence of a liver microsomal enzyme preparation (S-9 mix) from male rats pretreated with Aroclor 1254 at 320 µg without S-9 mix, at 80 µg an increased number of revertants was noted. Only a doubling of the revertant number was observed in presence of S-9 mix at 1280 µg. A toxic effect was noted at 5120 µg with S-9 mix and at 1280 µg and 5120 µg without S-9 mix.

A toxic effect was also observed with the strains TA 1537 at 5120 µg of product in presence and absence of S-9 mix and with the strain TA 100 at 5120 µg only in absence of S-9 mix.

	With S9
	TA 98	TA 100	TA 1535	TA 1537
Spontaneous reversion	58 (SD = 1)	167 (SD = 4)	15 (SD = 5)	9 (SD = 2)
Control DMSO	47 (SD = 12)	127 (SD = 9)	16 (SD = 3)	11 (SD = 2)
Positive control	2367 (SD = 90)	2740 (SD = 47)	769 (SD = 43)	283 (SD = 36)
Test substance (µg)
20.0	41 (SD = 5)	126 (SD = 11)	16 (SD = 2)	13 (SD = 3)
80.0	55 (SD = 12)	137 (SD = 29)	11 (SD = 1)	15 (SD = 3)
320	51 (SD = 8)	141 (SD = 2)	17 (SD = 3)	15 (SD = 3)
1280.0	115 (SD = 11)	127 (SD = 14)	14 (SD = 3)	13 (SD = 3)
5120.0	<0>	133 (SD = 9)	27 (SD = 2)	<0>
	Without S9
	TA 98	TA 100	TA 1535	TA 1537
Spontaneous reversion	24 (SD = 5)	99 (SD = 14)	21 (SD = 5)	6 (SD = 2)
Control DMSO	21 (SD = 1)	97 (SD = 6)	22 (SD = 4)	3 (SD = 1)
Positive control	756 (SD = 108)	2597 (SD = 116)	2257 (SD = 110)	291 (SD = 8)
Test substance (µg)
20.0	24 (SD = 2)	105 (SD = 9)	26 (SD = 5)	7 (SD = 1)
80.0	46 (SD = 5)	109 (SD = 4)	19 (SD = 4)	6 (SD = 2)
320	77 (SD = 9)	152 (SD = 15)	21 (SD = 7)	14 (SD = 4)
1280.0	0 (SD = 0)	158 (SD = 7)	16 (SD = 4)	6 (SD = 2)
5120.0	<0>	<0>	13 (SD = 1)	<0>

Conclusions:

Interpretation of results:
positive.

A mutagenic effect was observed with the strain TA 98 (with and without metabolic activation).

Executive summary:

The substance was tested with the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 at concentrations from 20 to 5120 µg per Petri dish both in the presence and absence of metabolic activation.

Under the experimental conditions defined in the protocol, and employing a doubling of the spontaneous reversion rate and dose-effect relationship as criteria of mutagenicity, the product was mutagenic for Salmonella typhimurium strain TA 98. No mutagenic effect was noted with the strains TA 1535, TA 1537 and TA 100.

With the strain TA 98 the evidence for mutagenicity existed only in the absence of a liver microsomal enzyme preparation (S-9 mix) from male rats pretreated at 320 µg without S-9 mix, at 80 µg an increased number of revertants was noted. Only a doubling of the revertant number was observed in presence of S-9 mix at 1280 µg. A toxic effect was noted at 5120 µg with S-9 mix and at 1280 µg and 5120 µg without S-9 mix.

A toxic effect was also observed with the strains TA 1537 at 5120 µg of product in presence and absence of S-9 mix and with the strain TA 100 at 5120 µg only in absence of S-9 mix.

 

Conclusion

From the results of Ames test, it appears that the test substance presents only a very slight mutagenic effect in this system. This effect is observed only with one tester strain (TA 98). This effect is markedly diminished when liver microsomial enzymes are added to the preparation suggesting that the compound might be detoxified in vivo.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Read-across - OECD TG 474 - key study - KS2: negative. 

Read-across - OECD TG 489 - Comet assay TPE. 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

not yet defined

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Justification for type of information:

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out: analogue substance 01

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION
- Available GLP studies: not available.
- Available non-GLP studies: not available.
- Historical human data: not available.
- (Q)SAR: not available.
- Weight of evidence: not available.
- Grouping and read-across:
• In vitro gene mutation study in bacteria (Ames test) – OECD TG 471 – RA on analogue substance 03.
• In vitro gene mutation study in bacteria (Ames test) – OECD TG 471 – RA on analogue substance 03.
• In vitro gene mutation study in bacteria (Ames test) – OECD TG 471 – NR deficient strains – RA on analogue substance 01.
• In vivo mammalian somatic cell study: cytogenicity/erythrocyte micronucleus – OECD TG 474 – RA on analogue substance 03.

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
Under Annex VIII Section 8.4., column 2 of REACH, further mutagenicity studies must be considered in case of a positive result in an in vitro gene mutation study in bacteria.
Guidance on information requirements R7a, section 7.7.6 (2017), states that regarding Annex VIII, when both the mammalian cell tests are negative but there was a positive result in the bacterial test, it will be necessary to decide whether any further testing is needed on a case-by-case basis. For example, suspicion that a unique positive response observed in the bacterial test was due to a specific bacterial metabolism of the test substance could be explored further by investigation in vitro. Alternatively, an in vivo test may be required.
The present dossier contains positive results for the in vitro gene mutation study in bacteria, following OECD TG 471 and conducted on analogue substance 03, which raises the concern for gene mutation.
As presented in the attached document regarding genotoxicity, a fist attempt on elucidating the effect of mechanism of the nitroreductase in the positivity of the Ames test was evaluated on the analogue substance 01. The analogue substance was tested in a modified Ames test following OECD TG 471 with NR deficient strains, namely TA 98 and TA 100, in a Prival method. The results really support the major role played by the nitroreductase and a great decrease on the number of revertants with the respect to the control was obtained for TA 98NR and TA 100NR. However, the modified Ames test cannot be finalised as totally negative.
Annex VIII, Column 2 requires the registrant to consider appropriate mutagenicity in vivo studies already at the Annex VIII tonnage level, in cases where positive results in genotoxicity studies have been obtained, which involves studies mentioned in Annex IX (as first step OECD TG 474, Mammalian Erythrocyte Micronucleus test, OECD TG 488, Transgenic Rodent Mutation Assay, OECD TG 489, in vivo Mammalian Alkaline Comet Assay and OECD TG 486, Unscheduled DNA Synthesis).

CONSIDERATIONS ON THE STUDIES INSERTED IN THE PRESENT DOSSIER AND EXPERT ASSESSMENT ON TESTING PROPOSAL
In the present dossier, an OECD TG 474 (Mammalian Erythrocyte micronucleus test) in vivo study is available on the analogue substance 03, with negative results, which is adequate to cover the chromosomal aberration potential of the substance.
However, to completely assess the gene mutation properties of the substance in different tissues of the animal, a Comet Assay, OECD TG 489, on the analogue substance 01 is presented as testing proposal.
OECD TG 489 allows to measure DNA strand breaks, that may result from direct interactions with DNA, alkali labile sites or as a consequence of incomplete excision repair. Therefore, the alkaline comet assay recognises primary DNA damage that would lead to gene mutations and/or chromosome aberrations, but will also detect DNA damage that may be effectively repaired or lead to cell death. The comet assay can be applied to almost every tissue of an animal from which single cell or nuclei suspensions can be made, including specific site of contact tissues.
OECD TG 488 is not considered the first choice for assessing the gene mutation in vivo for this substance, since preliminary data for gene mutation in vivo (OECD TG 474) already indicates negativity in the somatic bone marrow cells. A confirmation by the Comet assay performed over other tissues (and for azo dyes the intestinal tract is the site of major metabolism and dye/metabolites absorption) would be sufficient to assess the genotoxic potential of the substance.
Finally, as reported in literature, from the analysis of 91 chemicals with published data from Comet Assay and Transgenic rodent mutation assay (TGR), the comet assay appears to yield similar results to the TGR assay in liver and gastrointestinal tract (predominantly stomach and colon data) and, hence, can be confidently performed to confirm in vivo gene mutation activity in terms of genotoxicity in general.

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

GLP compliance:

yes

Type of assay:

mammalian comet assay

Sex:

not specified

Genotoxicity:

other: to be performed

Remarks on result:

other: the test is in read-across from a submitted testing proposal still under evaluation.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

other: read across from similar substance

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

no

Type of assay:

micronucleus assay

Species:

mouse

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Animals: non-consanguinous OF-1 albino mice.
- Source: mice originating from an SPF colony (IFFA-CREDO, L'Arbresle, France).
- Weight at study initiation: 25 g
- Housing: animals were housed 5 of the same sex per cage in Makrolon type III cages.
- Diet: ad libitum, aliment Rats-Souris Charles River, produced by U.A.R., Villemoisson/Orge, France.
- Water: ad libitum.
- Quarantine period: quarantine period of 1 week at test laboratory.

Route of administration:

oral: gavage

Details on exposure:

Mice receiving one intragastric intubation (1500 mg/kg) using 0.5 ml of a solution at 75 mg/ml per 25 g body weight.

Remarks:

Doses / Concentrations:
1500 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

Five male and five female mice per group. The groups were: three test item-treated animals, one negative control and one positive control.

Control animals:

other: Negative control (distilled water): 0.5 ml - animals sacrificed at 44 hours

Positive control(s):

N,N',N"-triethylenethiophosphoramide
- Source: Lederle Laboratories Ltd.
- Concentration administered: 20 mg/kg
- Necropsy: sacrificed at 44 hours

Tissues and cell types examined:

Mouse bone marrow smears.

Details of tissue and slide preparation:

Animals were sacrificed at regular intervals after treatment: one group at 20, one group at 44 and one group at 68 hours.

DETAILS OF SLIDE PREPARATION
After sacrifice of the animals the femurs were taken and broken open at one end. Bone marrow cells were suspended in foetal calf serum using a small syringe, and the cells were centrifuged at 120 x g for 5 minutes. The supernatant was removed with a Pasteur pipette, cells were spread on a microscope slide and the smears allowed drying in air. The following day smears were stained with Giemsa (1:6 in water) and mounted after drying with a coverslip.

ESTIMATION OF NUMBERS OF MICRONUCLEI
Two types of erythrocytes were observed in the bone marrow smears: normochromatic (mature red blood cells about to pass into the blood stream) and polychromatic (immature red blood cells). The latter are stained blue by Giemsa for around 24 h after the expulsion of the erythroblast nucleus: the colouration is probably due to traces of RNA remaining in these cells.

The proportion of polychromatic erythrocytes containing one or more micronuclei was compared with the total number of polychromatic erythrocytes, and statistical comparisons were made between these ratios for the different groups. A minimum of 500 polychromatic erythrocytes were counted per smear (two smears per animal).

In each smear an evaluation was made of the numbers of nucleated cells and the two types of erythrocytes (normochromatics and polychromatics) were counted up to a total of 2000 erythrocytes per animal. This was done in order to gain information on the mode of action of the test compound in bone marrow cells, and also to identify possible artifacts.
All slides were given coded labels and were microscopically analysed without knowledge of their treatment groups.

Statistics:

A complete statistical analysis using BMPD computer programme 7D was performed.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE FINDING
First a preliminary range finding test was carried out by treating three groups of three male mice at concentrations of 3000, 2000 and 1000 mg/kg, the dose of 3000 mg/kg being the limit of solubility of the compound. Only one death occurred at the dose of 3000 mg/kg and a mouse was in a poor state at the dose of 2000 mg/kg. Therefore a dose of 1500 mg/kg was chosen to perform the micronucleus test.

GENERAL APPEARANCE OF THE SMEARS
At low magnification of the microscope no noticeable differences in bone marrow nucleated cells were observed between animals treated with the test item and negative controls.
In the positive control group (Thio-TEPA) decreased numbers of bone marrow nucleated cells were noted.

TEST RESULTS
There was no statistically significant increase in the number of micronucleated polychromatic erythrocytes in animals exposed to 1500 mg/kg compared to negative control animals.
In animals treated with positive control there was a statistically significant increased number of micronucleated cells. The ratio of polychromatic to normochromatic erythrocytes was markedly decreased in mice treated with the positive control.
The only difference for this ratio observed between groups is a decrease in animals receiving the test item after 68 h compared to those sacrificed after 20 h. There is no statistically significant difference between controls and animals treated with the test item.

NUMBERS OF OBSERVED MICRONUCLEI AND RATIO OF POLYCHROMATIC TO NORMOCHROMATIC ERYTHROCYTES:

 

The percentage of polychromatic erythrocytes carrying one or more micronuclei, compared with the total number of polychromatic erythrocytes, was as follows:

Group	Dose mg/kg	Time of sacrifice	Micronucleous erythrocytes (%)
			Mean	S.D.
Negative control	0	44	0.32	0.17
Test material	1500	20	0.38	0.15
Test material	1500	44	0.4	0.16
Test material	1500	68	0.24	0.14
Positive control	20	44	21.3	8.25

S.D. = Standard Deviation

 

The proportion of polychromatic erythrocytes compared with the total number of erythrocytes was as follows:

Group	Dose mg/kg	Time of sacrifice	Polychromatic erythrocytes (%)
			Mean	S.D.
Negative control	0	44	58.1	6.3
Test material	1500	20	62.5	5.3
Test material	1500	44	55.3	8.9
Test material	1500	68	47.5	11.2
Positive control	20	44	13.8	7.0

S.D. = Standard Deviation

Conclusions:

Interpretation of results: negative.
No mutagenic effect was observed in bone marrow smears.

Executive summary:

The test compound was assayed for mutagenicity using the micronucleus test (OECD and EEC protocols). The compound was administered orally to mice at a concentration of 1500 mg/kg of a solution at the maximal concentration of 75 mg/ml.

No mutagenic effect was observed in bone marrow smears taken 20, 44 and 68 h after administration of the test substance. A positive control (Thio-TEPA) administered at a concentration of 20 mg/kg showed pronounced evidence of mutagenicity 44 h after administration. Thio-TEPA shows that the strain of mouse used is sensitive to mutagens and that such products can be detected using the micronucleus test.

 

Conclusion:

No mutagenic effect was observed in bone marrow smears.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

For further details refer to the attached document on genotoxicity assessment.

Justification for classification or non-classification

Classification for mutagenicity is warranted for substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans:

The classification in Category 2 is based on:

- Positive evidence obtained from experiments in mammals and/or in some cases from in vitro experiments, obtained from:

• Somatic cell mutagenicity tests in vivo, in mammals; or


• Other in vivo somatic cell genotoxicity tests which are supported by positive results from in vitro mutagenicity assays.

 

A new classification will be applied when results of the "In vivo" Comet assay on analogue substance 01 will be available.