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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 February 2018 - 22 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The test was initiated upon request of ECHA (decision number TPE-D-2114376203-54-01/F).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian comet assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Remarks:
Clear colorless liquid

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
Rat was selected as recommended in the guideline. The Crl:CD(SD) strain was selected, because BSRC has abundant usage experience and a large historical database on this strain.
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Age at study initiation: 8 weeks
- Weight at study initiation: 283 to 309 g
- Assigned to test groups randomly: yes
- Fasting period before study: No
- Housing: 2 rats/cage
- Diet: free access to commercial diet (CRF-1, lot No. 171024, Oriental Yeast)
- Water: free access to tap water from water bottles
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.8-23.2
- Humidity (%): 47-62
- Air changes (per hr): 12 or more
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 7 March 2018 - 8 March 2018

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Solvent used: corn oil
- Concentration of test material in vehicle: 25, 50 and 100 mg/mL
- Amount of vehicle: 10 mL/kg bw
- Lot no.: SAG4916 (Wako Pure Chemical Industries, Ltd.)
- The test substance is reported to be stable in the vehicle (corn oil) at room temperature under normal laboratory light conditions for at least 4 hours.
Details on exposure:
The individual dosing volume (mL) were calculated on the basis of the body weight measured at the time of assignment to dose groups.
Duration of treatment / exposure:
Two consecutive days
Frequency of treatment:
Once daily
Post exposure period:
3 hours
Doses / concentrationsopen allclose all
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
6 males were dosed, 5 males analysed
Control animals:
yes, concurrent vehicle
Positive control(s):
Ethyl methanesulfonate (500 mg) was dissolved in 25 mL of water for injection to prepare a 20 mg/mL solution (dose of 200 mg/kg bw). This positive control solution was prepared just before administration.

Examinations

Tissues and cell types examined:
Stomach tissue
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
In dose range-finding study in rats treated with 0, 222, 667 or 2000 mg/kg of test substance in corn oil, decrease in locomotor activity, salivation and diarrhea were observed in the 667 mg/kg group, and decrease in locomotor activity, salivation, loose stool, diarrhea and soiled fur were observed in the 2000 mg/kg bw group. No clinical signs of toxicity were observed in the 222 mg/kg bw group. The body weight of 667 and 2000 mg/kg bw groups decreased. In the gross observation, edema of the forestomach was observed in the 2000 mg/kg bw group. Therefore, 1000 mg/kg, which was considered to be the maximum tolerated dose, was selected as the highest dosage level and a total of 3 dosage levels, 250, 500 and 1000 mg/kg bw, were selected in the present study.

TREATMENT AND SAMPLING TIMES:
The oral route that was assumed to be one of the exposure routes for the human was selected as an administration route of the vehicle and the test substance formulations.

DETAILS OF SLIDE PREPARATION:
The stomachs were macroscopically examined at necropsy, and gross findings were recorded including the affected portion, color, size. Any fouling (blood, etc.) was rinsed off using homogenizing buffer. The forestomach was removed and discarded. The glandular section of the stomach was cut open and rinsed with the homogenizing buffer. After collecting a part of the glandular stomach for pathological specimen, the remaining glandular stomach was incubated in cooled homogenizing buffer for 15 to 30 minutes. After incubation, the surface epithelium was gently scraped several times using a blade and discarded. In a dish containing homogenizing buffer, the stomach epithelium was scraped several times with the blade to release the cells. The cell suspension was put into a tube. After the appropriate amount of the supernatant is discarded by centrifugation (800 rpm, 5 minutes), the cells were re-suspended in the remaining supernatant and 10 μL of the cell suspension was put into a microtube.
A superfrosted glass slide was pre-coated with 1.0% agarose gel. Ninety microliters of 0.5% low-melting agarose gel was added to a microtube. After mixing, 90 μL of the cell-agarose mixture was placed on the pre-coated slide and covered with a non-coated superfrosted glass slide. After the agarose solidified, the covering glass slide was removed. Another layer of 0.5% low-melting agarose (90 μL) was added in the same manner.
Each slide was placed in lysing solution and left overnight under the refrigerated and light-protected conditions. Three slides (2 slides for evaluation, 1 for spare) per organ were prepared.

METHOD OF ANALYSIS:
One hundred fifty cells (75 cells per slide), i.e. 750 cells per group (5 animals), were analyzed. Hedgehogs were excluded from comet image analysis. Separately, 150 cells per animal (75 cells per slide) were analyzed using the fluorescence microscope (×200), and the number of hedgehogs was counted.
The percentage of DNA in the tail relative to the total (% tail DNA: Tail % intensity) was used as an indicator for DNA damage. The median % tail DNA for each slide (slide value) was determined, and the mean of the slide values for each animal (animal value) was calculated.
Evaluation criteria:
Interpretation of the results:
a) At least one of the test substance-treated groups exhibits a statistically significant increase of % tail DNA compared with the negative control.
b) A significant dose dependence in the % tail DNA in the test substance-treated groups is present.
c) Any of the % tail DNA (mean of animal values) in the test substance-treated group is outside the distribution (mean±3SD) of the test facility’s historical data of the negative control group.
If all of the above criteria were met, the test result was considered to be positive. In addition, the biological relevance of the results was taken into consideration for the final judgment.

Validity of the study:
a) The mean % tail DNA in the negative control group should be within the acceptable ranges (mean±3SD) calculated from the test facility’s historical data.
b) The mean % tail DNA in the positive control group should be increased with a statistically significant difference compared with that in the negative control group. The study was considered to be successfully performed because the above criteria were met.
Statistics:
Each animal value of % tail DNA was logarithmically transformed prior to statistical analysis. The % tail DNA (mean of animal values) was analyzed by Dunnett’s multiple comparison test (two-sided, significant level of 0.05) between the negative control group and each test substance-treated group. Since no significant difference was observed, the dose dependence (trend) was not analyzed. For the comparison between the negative control group and positive control group, the % tail DNA (mean of animal values) was analyzed by Aspin-Welch’s t test (one-sided, significant level of 0.025).

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Decreased body weights, clinical signs and local effects on forestomach were seen at 500 and 1000 mg/kg bw.
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Mortality
No mortality occurred.

Gross necropsy
In the 500 and 1000 mg/kg bw treatment groups, edema in the forestomach was observed in 2 and 3 rats, respectively. There were no macroscopic findings in the 250 mg/kg bw group.

Body weight and general condition
The body weights of animals in 500 and 1000 mg/kg bw groups were decreased compared to control animals (mean weight loss of -7g, -17g, -32g for the groups dosed with 250, 500 and 1000 mg/kg bw versus a mean weight loss of -5g for the controls). No deaths were observed in any groups. In the 250 mg/kg group, loose stool was observed in two animals. In the 500 mg/kg bw and 1000 mg/kg bw groups, loose stool, soiled fur, decrease in locomotor activity, or diarrhea were observed in several animals.

DNA damage
In the negative control group, the mean % tail DNA was 3.87, which was within the acceptable ranges calculated from the test facility’s historical data.
The mean % tail DNA was 3.42, 3.63 and 3.83 for the NBDI-treated groups at 250, 500 and 1000 mg/kg bw, respectively. No statistically significant increase was observed as compared with the negative control group.
The frequencies of hedgehogs were 0.3, 0.7 and 0.4% for the NBDI-treated groups at 250, 500 and 1000 mg/kg bw, respectively, and no apparent increase was observed in any of the treatment groups as compared with the negative control group (1.6%).
The mean % tail DNA in the positive control group was 27.86 with a significant increase, indicative of a valid response.

Applicant's summary and conclusion

Conclusions:
In an alkaline Comet assay performed according to OECD guideline 489 and GLP principles, NBDI did not induce DNA damage in stomach cells when tested up to a maximum oral dose of 1000 mg/kg bw in rats.
Executive summary:

An in vivo alkaline comet assay was conducted according to OECD guideline 489 and GLP principles using Crl:CD(SD) male rats to assess the potential of NBDI to induce DNA damage. Based on a dose range finding study, the maximum tolerated dose was concluded to be 1000 mg/kg bw/day, therefore the assay was performed with three dose levels 250, 500 and 1000 mg/kg bw.

In the main study, decreased body weights, clinical signs and local effects on forestomach were seen at 500 and 1000 mg/kg bw. No statistically significant increase in the % tail DNA was observed in the NBDI-treated groups as compared with the negative control group. In addition, the % tail DNA were within the acceptable ranges (mean±3SD) calculated from this test facility’s historical data of the negative control group.

The % tail DNA in the negative control group were within the acceptable ranges calculated from this test facility’s historical data, and the % tail DNA in the positive control group was increased with a statistically significant difference compared with that in the negative control group. These results supported the validity of this study. It is therefore concluded that NBDI did not induce DNA damage in the stomach of rats under the conditions employed in this study.