16,17-dimethoxyviolanthrene-5,10-dione

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance did not induce any genotoxic effects in the studies conducted

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:

1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

only four strains used

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella typhimurium histidine (his) reversion system

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix (Rat liver enzymes)

Test concentrations with justification for top dose:

0, 20, 100, 500, 2500, 5000 and 10000 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

other: N-methyl-N'-nitro-N-nitroso-guanidine (MNNG) - TA 100 And TA 1535; 4-nitro-o-phenylenediamine - TA 98

Remarks:

without S9-mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9-mix

Details on test system and experimental conditions:

The test substance was tested in the standard plate test with and without S-9 mix in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 at

I. 0, 20, 100, 500, 2500, 5000 and 10000 µg/plate
II. 0, 100, 500, 2500, 5000 and 10000 µg/plate


The test substance was dissolved in DMSO and every concentration was tested in triplicate.

Evaluation criteria:

- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results.

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative 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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Incomplete solubility of the test substance in DMSO from 500 µg/plate onwards

Conclusions:

The test item was not mutagenic in the bacteria reverse mutation assay

Executive summary:

The test item was tested in the standard plate incorporation test in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 at concentrations from 20 to 10000 µg/plate both in the presence and absence of metabolic activation. An increase in the number of revertant colonies could not be observed up to the highest dose of 10000 µg/plate. Vat Green 1, purified is thus not mutagenic in the Ames test under the experimental conditions chosen.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January - March 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

The test item VatGreen 1 was examined for mutagenic activity by assaying for the induction of 6-
thioguanine resistant mutants in Chinese hamster V79 cells after in vitro treatment. 6-thioguanine can be
metabolised by the enzyme hypoxanthine-guaninphosphoribosyl-transferase (HPRT) into
nucleotides, which are used in nucleic acid synthesis resulting in the death of HPRT-competent cells. HPRTdeficient
cells, which are presumed to arise through mutations in the HPRT gene, cannot metabolise 6-thioguanine
and thus survive and grow in its presence.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Type and identity of media: EMEM medium supplemented with 10% Foetal Calf Serum (EMEM
complete)
- Properly maintained: yes; permanent stock of V79 cells are stored in liquid nitrogen and
subcoltures are prepared from the frozen stocks for experimental use.
- Periodically checked for Mycoplasma contamination: yes
- The karyotype, generation time, plating efficiency and mutation rates (spontaneous and induced)
have been checked in this laboratory.
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 tissue fraction: Species: Rat Strain: Sprague Dawley Tissue: Liver Inducing Agents: Phenobarbital – 5,6-Benzoflavone Producer: MOLTOX, Molecular Toxicology, Inc. Batch Number 3512

Test concentrations with justification for top dose:

A preliminary cytotoxicity assay was performed at the following dose levels: 100, 50.0, 25.0, 12.5, 6.25, 3.13, 1.56, 0.781 and 0.391 µg/mL
Two independent assays for mutation to 6-thioguanine resistance were performed using dose levels:
Main Assay I (-/+S9): 50.0, 25.0, 12.5, 6.25 and 3.13 μg/mL
Main Assay II (-S9): 50.0, 31.3, 19.6, 12.2 and 7.64 μg/mL
Main Assay II (+S9): 80.0, 50.0, 31.3, 19.6 and 12.2 μg/mL

Vehicle / solvent:

Test item solutions were prepared using dimethylsulfoxide (DMSO)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

A preliminary cytotoxicity test was undertaken in order to select appropriate dose levels for the mutation assays. In this test a wide range of dose levels of the test item was used. cell cultures were treated using the same treatment conditions as the mutation assays, and the survival of the cells was subsequently determined. Treatments were performed both in the absence and presence of S9 metabolism; a single culture was used at each test point and positive controls were not included.
Two Mutation Assays were performed including negative and positive controls, in the absence and presence of S9 metabolising system. Duplicate cultures were prepared at each test point, with the exception of the positive controls which were prepared in a single culture. On the day before the experiment, sufficient numbers of 75 cm^2 flasks were inoculated with 2 million freshly trypsinised V79 cells from a common pool. The cells were allowed to attach overnight prior to treatment. Following treatment, the cultures were incubated at 37°C for three hours. At the end of the incubation period, the treatment medium was removed and the cell monolayers were washed with PBS. Fresh complete medium was added to the flasks which were then returned to the incubator at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) to allow for expression of the mutant phenotype.
Determination of survival: The following day, the cultures were trypsinised and an aliquot was diluted and plated to estimate the viability of the cells.
Subculturing: At Day 1 a number of cells was replated in order to maintain the treated cell populations. On Day 3, the cell populations were subcultured in order to maintain them in exponential growth. When Day 9 is used as expression time, subculturing was performed also at Day 6.
Determination of mutant frequency: A single expression time was used for each experiment: Day 6 in Main Assay I and Day 9 in Main Assay II. At the expression time, each culture was trypsinised, resuspended in complete medium and counted by microscopy. After dilution, an estimated 1 x 10^5 cells were plated in each of five 100 mm tissue culture petri dishes containing medium supplemented with 6-thioguanine. These plates were subsequently stained with Giemsa solutions and scored for the presence of mutants. After dilution, an estimated 200 cells were plated in each of three 60 mm tissue culture petri dishes. These plates were used to estimate Plating Efficiency (P.E.).

Evaluation criteria:

For a test item to be considered mutagenic in this assay, it is required that:
- There is a five-fold (or more) increase in mutation frequency compared with the solvent controls, over two consecutive doses of the test item. If only the highest practicable dose level (or the highest dose level not to cause unacceptable toxicity) gives such an increase, then a single treatment-level will suffice.
- There must be evidence for a dose-relation (i.e. statistically significant effect in the ANOVA analysis).

Statistics:

The results of these experiments were subjected to an Analysis of Variance in which the effect of replicate culture and dose level in explaining the observed variation was examined. For each experiment, the individual mutation frequency values at each test point were transformed to induce homogeneous variance and normal distribution.
The appropriate transformation was estimated using the procedure of Snee and Irr (1981), and was found to be y = (x + a)^b where a = 0 and b = 0.275. A two way analysis of variance was performed (without interaction) fitting to two factors:
- Replicate culture: to identify differences between the replicate cultures treated.
- Dose level: to identify dose-related increases (or decreases) in response, after allowing for the effects of replicate cultures and expression time.
The analysis was performed separately with the sets of data obtained in the absence and presence of S9 metabolism.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Survival after treatment:
No relevant toxicity was observed at any concentration tested, in any experiment, in the absence or presence of S9 metabolism

Mutation results:
No relevant increases over the spontaneous mutation frequency were observed in any experiment, at any treatment level in the absence or presence of S9 metabolic activation. In Main Assay II, in the absence of S9 metabolic activation, a five-fold increase in mutation frequency was observed at an intermediate dose level. However, heterogeneity was noticed between replicate cultures, mutation frequencies of both replicates fell within the historical control range and the effect was not observed inMain Assay I. Hence, the observed increase was considered an incidental event not related to the action of the test item and of no biological significance. Analysis of variance indicated that dose level and replicate culture were not significant factors in explaining the observed variation in the data, in both experiments in the absence of S9 metabolism and in Main Assay II in its presence. In Main Assay I in the presence of S9 metabolism, dose level was a significant factor (p<0.01%) since mutation frequencies were greater at higher concentrations.
However, the induced mutation frequency did not reach five-fold the concurrent negative control, at any concentration tested. Moreover all mutation frequencies fell within the historical control range. Therefore the statistically significant linear trend was considered to be attributable to an incidental event without any biological relevance.

Conclusions:

Vat Green 1 does not induce mutation in Chinese hamster V79 cells after in vitro treatment, in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Executive summary:

The test item Vat Green 1 was examined for mutagenic activity by assaying for the induction of 6-thioguanine resistant mutants in Chinese hamster V79 cells after in vitro treatment. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. Test item suspensions/solutions were prepared using dimethylsulfoxide (DMSO). A preliminary cytotoxicity assay was performed. Based on solubility features, the test item was assayed, in the absence and presence of S9 metabolism, at a maximum dose level of 100 µg/mL and at a wide range of lower dose levels: 50.0, 25.0, 12.5, 6.25, 3.13, 1.56, 0.781 and 0.391 µg/mL. By the end of treatment, precipitation of the test item was noted at the two highest dose levels. No toxicity was observed at any concentration tested, in the absence or presence of S9 metabolic activation. Two independent assays for mutation to 6-thioguanine resistance were performed using dose levels described in the following table:

Main Assay I (-/+S9): 50.0, 25.0, 12.5, 6.25 and 3.13 μg/mL

Main Assay II (-S9): 50.0, 31.3, 19.6, 12.2 and 7.64 μg/mL

Main Assay II (+S9): 80.0, 50.0, 31.3, 19.6 and 12.2 μg/mL

Selection of dose levels used in Main Assay I was performed taking into account precipitation observed in the preliminary cytotoxicity assay. The dose range used in Main Assay II was modified to focus on the highest concentrations that could be tested. No relevant increases in mutant numbers or relevant five-fold increases in mutant frequency were observed following treatment with the test item at any dose level, in the absence or presence of S9 metabolism. Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. Marked increases were obtained with the positive control treatments, indicating the correct functioning of the assay system. It is concluded that Vat Green 1 does not induce gene mutation in Chinese hamster V79 cells after in vitro treatment, in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The test substance did not induce genotoxic effects

Link to relevant study records

Reference

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 August - Date of Study Director’s signature on this report

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

other: Mammalian Erythrocyte Micronucleus Test

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS srl., San Pietro al Natisone (UD), Italy
- Age at study initiation: 6 to 7 weeks
- Weight at study initiation: 187.8-206.4 g for males and 150.2-171.7 g for females
- Assigned to test groups randomly: yes, by computerised stratified randomisation to give approximately equal initial group mean body weights
- Fasting period before study:
- Housing: 5 of one sex to a cage - main groups from arrival to pairing; main group males after pairing; recovery groups throughout the entire study period
one male to one female- main groups during pairing
single - main group females after pairing
- Diet (ad libitum): 4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy
- Water (ad libitum): tap water
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): approximately 15 to 20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

sesame oil

Details on exposure:

The test item was administered orally by gavage at a dose volume of 5 mL/kg body weight.
Control animals received the vehicle alone at the same dose volume. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.

Duration of treatment / exposure:

Males:
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing, through the mating period and thereafter at least untilthe minimum total dosing period of 28 days had been completed including the day before necropsy. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.
Females:
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until at least up to, and including, Day 3 post partum or the day before sacrifice. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.
During the gestation period, dose volumes were calculated according to individual body weight on Days 0, 7, 14 and 20 post coitum and then on Day 1 post partum. Thereafter individual dose volumes remained constant.
Positive Control group (Group 7)
The Mitomycin-C (positive control) was administered once by intraperitoneal injection at the dose volume of 10 mL/kg body weight. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.

Frequency of treatment:

once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

62.5 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males and 5 females of the main groups randomly selected
In the absence of substantial inter-sex differences of toxicity, micronucleus testing in a single sex was sufficient. Therefore, slides from only male animals were analysed for micronucleus induction.

Control animals:

yes, concurrent vehicle

Positive control(s):

For genotoxicity endpoint, a satellite positive control group (5 animalsl/sex) was added.

Tissues and cell types examined:

Bone marrow from one femur.

Details of tissue and slide preparation:

Slide preparation and evaluation:
Samples of bone marrow were collected between 18 and 24 hours following the final treatment and approximately 48 hours following the second last treatment from 5 males and 5 females of the main groups randomly selected. Samples of bone marrow were also collected approximately 24 hours after single treatment from all animals of the positive control group (Group 7). One femur of each animal was removed and bone marrow cells obtained by flushing with foetal calf serum. The cells were centrifuged and a concentrated suspension prepared to make smears on slides. These slides were air-dried, fixed with methanol and then stained with haematoxylin and eosin solutions and mounted with Eukitt. Three slides were made from each animal.

Only slides from male animals were examined. The slides were randomly coded by a person not involved in the subsequent microscope scoring and examined under low power to select one or more slides from each animal according to staining and quality of smears. Four thousand polichromatic erythrocytes (PCEs) per animal were examined for the presence of micronuclei at high power (x 100 objective, oil immersion). At the same time, the numbers of normal and micronucleated normochromatic erythrocytes (NCEs) were also recorded.

Evaluation criteria:

Acceptance criteria:
The assay was considered valid if the following criteria were met:
– The incidence of micronucleated PCEs of the vehicle control group fell within the
historical negative control range.
– The positive control item results fell within the historical control range and were significantly increased, at statistical analysis, when compared with the concurrent negative control.
– 5 animals per group were available for slide analysis.

Evaluation criteria:
The test item was considered to induce micronuclei if a statistically significant increase in the micronucleus incidence of polychromatic erythrocytes (at P<0.05) was observed in any treatment group and a dose-effect relationship was demonstrated.
Where statistically significant increases in the incidence of micronucleated PCEs were observed, but all results were inside the distribution of negative controlvalues within this laboratory, then historical control data were used to demonstrate that these increases did not have any biological significance.

Statistics:

Only counts obtained from polychromatic cells were subjected to statistical analysis and the original observations (and not micronucleus frequencies per 1000 cells) were used. The variation between individual animals within each treatment group was assessed by χ2 calculation. In case of no significant heterogeneity within either group, the χ2 test was employed to compare treated groups with the vehicle control. If at least one of the groups was not homogeneous, the variance ratio (F) value was calculated from the between-group and within-group χ2 values to show the significance of any difference between treated and vehicle control groups. In addition, a test for a linear trend (Snedecor and Cochran) was performed in order to evaluate dose-effect relationship.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Proof of absorption
Individual overnight urine samples were collected from males. The absorbance of urine samples were analysed with a spectrophotometer (UV-visible spectrophotometer) using a wavelength of 530 nm (max absorption for Vat Green 1) and 1 cm optical cell, for a qualitative measurement of Vat Green 1. The absorbance of urine samples was also read at 473 nm as additional measurement. The results showed no differences in the absorbance values between treated and control groups. The presence of dark precipitate in many urine samples from treated animals was recorded and considered as a proof of absorption, as the dye is not soluble in urine and is hence found as a precipitate after excreteion.

Conclusions:

On the basis of the results obtained, it is concluded that VAT GREEN 1 does not induce micronuclei in the polychromatic erythrocytes of treated rats, under the reported experimental conditions.

Executive summary:

The investigation for possible cytogenetic effects of Vat Green 1 was included into a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity screening test in rats, as the test item is not soluble in water or organic solvents.

In this test, no relevant differences in clinical signs were observed between male and female animals from the main and recovery groups. Based on these results, the genotoxicity assessment was performed including male animals only.

The presence of a dark precipitate in many urine samples collected from treated animals was considered a proof of absorption.

Incidence of micronucleated cells

Following treatment with the test item, no relevant increase in the number of micronucleated PCEs over the concurrent negative control was observed at any dose level. The incidences of micronucleated PCEs were comparable to historical control data for negative control animals.

A marked increase in the frequency of micronucleated PCEs was observed in the positive control group.

Bone marrow cell toxicity

The ratio of mature to immature erythrocytes and the proportion of immature erythrocytes among total erythrocytes were analysed to evaluate the bone marrow cell toxicity. Based on these results, no relevant inhibitory effect on erythropoietic cell division was observed at any dose level.

Validity of the assay

The incidence of micronucleated PCEs of the negative control group fell within the historical control range (95% confidence limit).

Statistically significant increases in the incidence of micronucleated PCEs over the negative control values were seen in the positive control group. The induced response was compatible with the historical control range, demonstrating the laboratory proficiency in the conduct of the test.

Five animals per group were available for micronucleus slide analysis.

Based on the stated criteria, the assay was therefore accepted as valid.

Analysis of results

No significant within-group heterogeneity was observed; hence thec² test was used to compare treated groups with the control group.

Following treatment with the test item, no statistically significant increase in the incidence of micronucleated PCEs over the negative control value was observed at any dose level. No significant dose-effect relationship was found after a trend test evaluation.

 

Dose level	Incidence in micronucleated PCEs			PCE/s(PCEs+NCEs)
[mg/kg/day]	Mean	SE	Range	[%] over the mean control value
0.00	0.6	0.1	0.5 - 0.8	100
62.5	0.6	0.1	0.5 - 0.8	100
250	0.6	0.1	0.3 - 1.0	96
1000	0.7	0.1	0.3 - 1.0	99
Mitomycin-C 2.00 mg/kg	9.3	0.7	7.0 - 11.0	91

Conclusion

On the basis of the results obtained, it is concluded that VAT GREEN 1 does not induce micronuclei in the polychromatic erythrocytes of treated rats, under the reported experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The test item was tested in the standard plate incorporation test in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 at concentrations from 20 to 10000 µg/plate both in the presence and absence of metabolic activation. An increase in the number of revertant colonies could not be observed up to the highest dose of 10000 µg/plate. Vat Green 1 is thus not mutagenic in the Ames test under the experimental conditions chosen.

The test item was examined for mutagenic activity by assaying for the induction of 6-thioguanine resistant mutants in Chinese hamster V79 cells after in vitro treatment. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. Test item suspensions/solutions were prepared using dimethylsulfoxide (DMSO). A preliminary cytotoxicity assay was performed. Based on solubility features, the test item was assayed, in the absence and presence of S9 metabolism, at a maximum dose level of 100 µg/mL and at a wide range of lower dose levels: 50.0, 25.0, 12.5, 6.25, 3.13, 1.56, 0.781 and 0.391 µg/mL. By the end of treatment, precipitation of the test item was noted at the two highest dose levels. No toxicity was observed at any concentration tested, in the absence or presence of S9 metabolic activation. Two independent assays for mutation to 6-thioguanine resistance were performed using dose levels described in the following table:

Main Assay I (-/+S9): 50.0, 25.0, 12.5, 6.25 and 3.13 μg/mL

Main Assay II (-S9): 50.0, 31.3, 19.6, 12.2 and 7.64 μg/mL

Main Assay II (+S9): 80.0, 50.0, 31.3, 19.6 and 12.2 μg/mL

Selection of dose levels used in Main Assay I was performed taking into account precipitation observed in the preliminary cytotoxicity assay. The dose range used in Main Assay II was modified to focus on the highest concentrations that could be tested. No relevant increases in mutant numbers or relevant five-fold increases in mutant frequency were observed following treatment with the test item at any dose level, in the absence or presence of S9 metabolism. Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. Marked increases were obtained with the positive control treatments, indicating the correct functioning of the assay system. It is concluded that Vat Green 1 does not induce gene mutation in Chinese hamster V79 cells after in vitro treatment, in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

The investigation for possible cytogenetic effects of Vat Green 1 was included into a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity screening test in rats, as the test item is not soluble in water or organic solvents.

In this test, no relevant differences in clinical signs were observed between male and female animals from the main and recovery groups. Based on these results, the genotoxicity assessment was performed including male animals only. The presence of a dark precipitate in many urine samples collected from treated animals was considered a proof of absorption.

Following treatment with the test item, no relevant increase in the number of micronucleated PCEs over the concurrent negative control was observed at any dose level. The incidences of micronucleated PCEs were comparable to historical control data for negative control animals. A marked increase in the frequency of micronucleated PCEs was observed in the positive control group.

The ratio of mature to immature erythrocytes and the proportion of immature erythrocytes among total erythrocytes were analysed to evaluate the bone marrow cell toxicity. Based on these results, no relevant inhibitory effect on erythropoietic cell division was observed at any dose level.

The incidence of micronucleated PCEs of the negative control group fell within the historical control range (95% confidence limit). Statistically significant increases in the incidence of micronucleated PCEs over the negative control values were seen in the positive control group. The induced response was compatible with the historical control range, demonstrating the laboratory proficiency in the conduct of the test. Five animals per group were available for micronucleus slide analysis. Based on the stated criteria, the assay was therefore accepted as valid.

No significant within-group heterogeneity was observed; hence thec² test was used to compare treated groups with the control group. Following treatment with the test item, no statistically significant increase in the incidence of micronucleated PCEs over the negative control value was observed at any dose level. No significant dose-effect relationship was found after a trend test evaluation.

 

Dose level	Incidence in micronucleated PCEs			PCE/s(PCEs+NCEs)
[mg/kg/day]	Mean	SE	Range	[%] over the mean control value
0.00	0.6	0.1	0.5 - 0.8	100
62.5	0.6	0.1	0.5 - 0.8	100
250	0.6	0.1	0.3 - 1.0	96
1000	0.7	0.1	0.3 - 1.0	99
Mitomycin-C 2.00 mg/kg	9.3	0.7	7.0 - 11.0	91

Conclusion

On the basis of the results obtained, it is concluded that Vat Green 1 does not induce micronuclei in the polychromatic erythrocytes of treated rats, under the reported experimental conditions.