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Toxicological information

Genetic toxicity: in vivo

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
other: Mammalian Erythrocyte Micronucleus Test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Name: Vat Green 1

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals 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

Administration / exposure

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
Doses / concentrationsopen allclose all
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):
- Positive control: mytomicin c
- Justification for choice of positive control(s): according to guideline
- Route of administration: intraperitoneal injection
- Doses / concentrations: 2.00 mg/kg bw (0.2 mg/mL; 10 mL/kg bw)

Examinations

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.

Results and discussion

Test results
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.

Applicant's summary and conclusion

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.