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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The study has been requested by the European Chemicals Agency (ECHA Decision SEV-D-2114325960-51-01/F.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Principles of method if other than guideline:
The objective of this study was to obtain information on the potential genotoxicity of 1,2-Dichlorobenzene when administered to Wistar rats at the maximum recommended dose in accordance with current regulatory guidelines, by measuring the increase in the number of micronucleated polychromatic erythrocytes per 4000 polychromatic erythrocytes in rat bone marrow and by measuring the increase in DNA strand breaks in bone marrow, liver, duodenum and stomach.
GLP compliance:
yes
Type of assay:
mammalian comet assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1,2-dichlorobenzene
EC Number:
202-425-9
EC Name:
1,2-dichlorobenzene
Cas Number:
95-50-1
Molecular formula:
C6H4Cl2
IUPAC Name:
1,2-dichlorobenzene
Specific details on test material used for the study:
Purity/Composition: 99.9%

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Wistar WI (Han) rats (SPF) were used as the test system. These rats are recommended by international guidelines (e.g. OECD, EC). The animals were provided by Charles River, Sulzfeld, Germany.
Sex:
male
Details on test animals or test system and environmental conditions:
Test System

The study plan of this type of study was reviewed and agreed by the Laboratory Animal Welfare Officer and the Ethical Committee of Charles River Den Bosch as required by the Dutch Act on Animal Experimentation (February 1997).
Wistar WI (Han) rats (SPF) were used as the test system. These rats are recommended by international guidelines (e.g. OECD, EC). The animals were provided by Charles River, Sulzfeld, Germany.
Young adult animals were selected (9-11 weeks old at the start of treatment). The total number of animals used in the dose-range finding study was 4 and in the main study 25. In the main study 5 male rats were treated per sampling time in each treatment group.
The body weights of the rats at the start of the treatment in the main study were within 20% of the sex mean. The mean body weights were 274.2 ± 9.3 g and the range 256 – 289 g. The rats were identified by a unique number on the tail written with a marker pen. The animals were allocated at random to the treatment groups.
The acclimatization period was at least 6 days before the start of treatment under laboratory conditions.
On arrival and at the start of the treatment, all animals were clinically examined to ensure selected animals were in a good state of health.

Husbandry

Environmental Conditions
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 19.3 to 20.2°C with an actual daily mean relative humidity of 47 to 70%. A 12 hour light/12 hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
Accommodation
Group housing of maximum 5 animals in labeled Macrolon cages (type MIV height 180 mm, length 600 mm and width 330 mm) containing sterilized sawdust as bedding material.
Diet
The animals had free access to pelleted rodent diet. Results of analyses for nutrients and contaminants of each batch were examined and archived.
Water
The animals had free access to tap-water.

Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Preparation of Test Item
No correction was made for the purity/composition of the test item.
A solubility test was performed based on visual assessment. The test item was dissolved corn oil. The specific gravity of corn oil is 0.9 g/ml. The density of corn oil was taken into account when preparing the formulations.
The test item concentrations were dosed within 3 hours after preparation.
Details on exposure:
The rats were dosed for three consecutive days (once daily) using an oral intubation of a maximum tolerated (high), an intermediate and a low dose of the test item. The rats were dosed twice with the positive control EMS (Ethyl Methane sulfonate) and once with CP (cyclophosphamide).
A limited quantity of food was supplied during the night before dosing (approximately 7 g/rat). The route of administration of the test item was selected taking into account the possible route of human exposure during manufacture, handling and use.
The first dose of the test item and vehicle was administered at t=0 h. The second and third dose were administered at approximately t=24 h and t=45 h, respectively. The positive control CP was administered once at t = 0 h and EMS was administered at t=24 and t=45. The animals were sacrificed at by abdominal aorta bleeding under isoflurane anesthesia at t=45h.
The dosing volume was 10 ml/kg.
1,2-Dichlorobenzene concentrations were used within 3 hours after preparation.
CP is the positive control for the MN assay, and EMS the positive control for the Comet assay.
Duration of treatment / exposure:
The animals were sacrificed by abdominal aorta bleeding under isoflurane anesthesia at t=45h.
Frequency of treatment:
The rats were dosed for three consecutive days (once daily).
Post exposure period:
None
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Vehicle
Dose / conc.:
125 mg/kg bw/day
Remarks:
1,2-dichlorobenzene
Dose / conc.:
250 mg/kg bw/day
Remarks:
1,2-dichlorobenzene
Dose / conc.:
500 mg/kg bw/day
Remarks:
1,2-dichlorobenzene
Dose / conc.:
200 mg/kg bw/day
Remarks:
EMS
Comet assay
No. of animals per sex per dose:
5 male animals/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
EMS (Ethyl Methane sulfonate)

Examinations

Tissues and cell types examined:
The objective of this study was to obtain information on the potential genotoxicity of 1,2-Dichlorobenzene when administered to Wistar rats at the maximum tolerated dose in accordance with current regulatory guidelines,by measuring the increase in DNA strand breaks in bone marrow, liver, duodenum and stomach.
Details of tissue and slide preparation:
Approximately 3-5 hours after the third treatment with the test item bone marrow was isolated for the micronucleus test. In addition bone marrow, liver, duodenum and stomach were collected/isolated and examined for DNA damage with the alkaline Comet assay.
The slides were examined with a fluorescence microscope connected to a Comet Assay IV image analysis system (Perceptive instruments Ltd, Suffolk, United Kingdom).
One hundred fifty Comets (50 comets of each replicate LMAgarose circle) were examined per sample. On a few slides it was not possible to score 50 comets per agarose circle, therefore the remaining number of comets were scored on an additional agarose circle.









Evaluation criteria:
Acceptability of the Comet Assay
The in vivo comet is considered acceptable if it meets the following criteria:
a) The percentage tail intensity of the solvent control should reasonably be within the laboratory historical control data range.
b) The positive control EMS should produce at least a statistically significant (one-sided, p < 0.05) increase in the percentage Tail Intensity compared to the negative control treated animals using the Student’s t-test.
Statistics:
A test item is considered as giving a positive result in the Comet assay if the following criteria are met:
a) A statistically significant (Dunnett’s test, one-sided, p < 0.05) dose-dependent increase (indication of strand breaks) in percentage Tail Intensity is detected.
A test item is considered as negative in the Comet assay (in a tissue) if the following criteria are met:
a) None of the tested concentrations show a statistically significant (Dunnett’s test, one-sided, p < 0.05) dose-dependent increase in percentage Tail Intensity.
All data was normally distributed thus no transformation (y = 1/y) of the data was necessary.
A trend test was performed for liver and stomach.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
other: No genotoxic effect was observed in the comet assay in bone marrow, duodenum and liver. In stomach, the result of the comet assay was equivocal.
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
In a dose-range finding study 4 animals (group A: 3 males and group B: 1 male) were dosed via oral gavage with 500 and 750 mg/kg body weight of the test item (group A and B, respectively). The animal dosed with 750 mg/ kg body weight showed generally treatment related clinical signs of severe intensity after dosing and didn’t recover before the next dosing. The clinical signs comprised of lethargy, ataxia, rough coat, hunched posture, ventral recumbancy, tremors and not all feed provided overnight was eaten. A dose of 750 mg/kg/day was therefore considered as too high as top dose for the main study.
Three males were dosed with 500 mg/kg body weight. All animals showed clear clinical signs. The effects were generally of slight intensity and comprised lethargy, rough coat, ataxia, hunched posture, ventral recumbency and not all feed provided overnight was eaten. A dose of 500 mg/kg/day was therefore considered as the maximum tolerated dose as defined in the genotoxicity OECD 489 and 474 guideline and selected as top dose for the main study.

RESULTS OF DEFINITIVE STUDY
None of the animals in the vehicle control group, positive control groups and group treated with 125 mg/kg 1,2-dichlorobenzene showed abnormal clinical signs with exception of two animals in the positive control group of the comet assay treated with 200 mg/kg EMS which had a rough coat after the second dosing.
Animals treated with 250 and 500 mg/kg 1,2-dichlorobenzene showed the following signs: lethargy, ataxia (500 mg/kg only), rough coat, hunched posture and not all feed provided overnight was eaten.

Comet Test:
The mean Tail Intensity in bone marrow, liver, duodenum and stomach was all well within the historical control data range. Moreover, the positive control EMS induced a statistically significant increase in the Tail Intensity in bone marrow, liver, duodenum and stomach. Overall it was concluded that the comet assay in bone marrow, liver, duodenum and stomach was valid.
No statistically significant increase in the mean Tail Intensity (%) was observed in bone marrow and duodenum of test item treated male animals compared to the vehicle treated animals. In addition, no statistically significant increase in the mean Tail Intensity (%) was observed in liver cells of 1,2-dichlorobenzene-treated male animals at a dose of 125 and 250 mg/kg/day compared to the vehicle treated animals. Only at 500 mg/kg a statistically significant (slight) increase in the Tail Intensity was observed (2.1-fold increase, p<0.05 Dunnett’s t test) from 2.35% in the vehicle control to 4.87% at 500 mg/kg. A trend test was performed and a statistically significant trend was observed (p<0.05, non-parametric trend analysis by contrast). In stomach cells the 125 and 250 mg/kg treatment group showed a significant increase in mean Tail Intensity (%) from 23.85% in the vehicle control to 57.99% at 125 mg/kg (2.0-fold increase) and to 56.72% (2.4-fold increase) at 250 mg/kg (p<0.05 Dunnett’s t test). At 500 mg/kg no statistically increase was observed. A trend test was performed and a statistically significant trend was observed (p<0.05, non-parametric trend analysis by contrast).

Histopathology
All rats were necropsied and the part of the liver, stomach and duodenum was fixed. Microscopic examination was performed on the liver and stomach of all animals since an increase in DNA damage was observed in these two organs after test item treatment.
Test item-related microscopic findings were present in the

Liver:
• Centrilobular necrosis was present in all 1,2-Dichlorobenzene treated groups in a dose dependent manner and up to marked degree.
Centrilobular mononuclear inflammatory cell infiltrate was present in all 1,2-Dichlorobenzene treated groups in a dose dependent manner and up to moderate degree.

Glandular stomach:
• Hypertrophy parietal cells was present in all 1,2-Dichlorobenzene treated groups in a dose dependent manner and up to slight degree.
Of these findings, the centrilobular necrosis in the liver is the only finding representing cell death and causing increased DNA damage.



Any other information on results incl. tables

•  No increase in DNA damage was observed in the comet assay in bone marrow, and duodenum of male rats up to a dose of 500 mg/kg (the maximum tolerated dose in accordance with current regulatory guidelines).

•  In liver cells a slight increase in DNA damage in the comet assay was observed at 500 mg/kg from 2.35% in the vehicle control to 4.87% at 500 mg/kg. Histopathology showed that at this dose centrilobular necrosis was present up to a marked     degree.  Therefore it can be concluded that the DNA damage observed at 500 mg/kg is caused by necrosis.  Moreover, the Tail Intensity of 4.87% is clearly with in the historical data control range of the comet assay in liver. Thus overall it can be concluded for liver that necrosis is present at a dose of 500 mg/kg but that no genotoxic effect in liver is observed up to a dose of 500 mg/kg (the maximum tolerated dose in accordance with current regulatory guidelines).

•  In stomach a statistical significant increase in DNA damage was observed at 125 and 250 mg/kg. But since no increase was observed at 500 mg/kg, no dose response was observed.  Histopathological examination showed no necrosis in stomach at any of the tested concentrations.  Thus DNA damage was observed after treatment but since this effect showed no dose response, the overall conclusion is equivocal and additional investigation in a comet assay in stomach is required.

•  Thus overall it can be concluded that no genotoxic effect was observed in the comet assay in bone marrow, duodenum and liver. In stomach, the result of the comet assay was equivocal.

Applicant's summary and conclusion

Conclusions:
Overall it can be concluded that no genotoxic effect was observed in the comet assay in bone marrow, duodenum and liver. In stomach, the result of the comet assay was equivocal.
Executive summary:

The objective of this study was to obtain information on the potential genotoxicity of 1,2-Dichlorobenzene when administered to Wistar rats at the maximum recommended dose in accordance with current regulatory guidelines, by measuring the increase in the number of micronucleated polychromatic erythrocytes per 4000 polychromatic erythrocytes in rat bone marrow and by measuring the increase in DNA strand breaks in bone marrow, liver, duodenum and stomach.

As a result

•  No increase in DNA damage was observed in the comet assay in bone marrow, and duodenum of male rats up to a dose of 500 mg/kg (the maximum tolerated dose in accordance with current regulatory guidelines).

•  In liver cells a slight increase in DNA damage in the comet assay was observed at 500 mg/kg from 2.35% in the vehicle control to 4.87% at 500 mg/kg. Histopathology showed that at this dose centrilobular necrosis was present up to a marked degree. Therefore it can be concluded that the DNA damage observed at 500 mg/kg is caused by necrosis. Moreover, the Tail Intensity of 4.87% is clearly with in the historical data control range of the comet assay in liver. Thus overall it can be concluded for liver that necrosis is present at a dose of 500 mg/kg but that no genotoxic effect in liver is observed up to a dose of 500 mg/kg (the maximum tolerated dose in accordance with current regulatory guidelines).

•  In stomach a statistical significant increase in DNA damage was observed at 125 and 250 mg/kg. But since no increase was observed at 500 mg/kg, no dose response was observed.  Histopathological examination showed no necrosis in stomach at any of the tested concentrations.  Thus DNA damage was observed after treatment but since this effect showed no dose response, the overall conclusion is equivocal.

Thus overall it can be concluded that no genotoxic effect was observed in the comet assay in bone marrow, duodenum and liver. In stomach, the result of the comet assay was equivocal.

Detailed discussion on glandular stomach:

The mean Tail Intensities in stomach cells are as follows:

   Mean Tail Intensity (%) (1)  S.D.
 Vehicle Control  23.85 6.93
 1,2-Dichlorobenzene 125 mg/kg  47.99 (2) 14.37
 1,2-Dichlorobenzene 250 mg/kg  56.72 (2) 7.10
 1,2-Dichlorobenzene 500 mg/kg  34.61 19.60
 EMS 200 mg/kg  97.32 (3) 0.68

(1)Five animals per treatment group.

(2) Statistically significant from the control group (Dunnett’s t test, p<0.05)

(3) Significantly different from corresponding control group (Students t test, p < 0.001).

The observations in the stomach are considered to be equivocal because:

1) No clear dose response is observed.

in stomach cells the 125 and 250 mg/kg treatment group showed a significant increase in mean Tail Intensity (%) from 23.85% in the vehicle control to 47.99% at 125 mg/kg (2.0-fold increase) and to 56.72% (2.4-fold increase) at 250 mg/kg (p<0.05 Dunnett’s t test). At 500 mg/kg a statistically non-significant 1.5-fold increase was observed.

2) the Tial Intensity reported is within historical control values.

The mean Tail Intensities of the low, mid and high dose group are within the historical control values and far below the historical positive control values observed with EMS.

Historical data Comet assay Negative control data:

  Stomach Tail Intensity (%) Males and Females
 Range  2.45 – 72.13
 Mean  35.94
 SD  19.88
 n  44

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed in 2012 – 2017

Historical data Comet assay (200 mg/kg EMS orally dosed for two consecutive days):

  Stomach Tail Intensity (%) Males and Females
Range 85.69 – 99.19
Mean 93.53
 SD  3.67
 n  42

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed in 2012 – 2017

3) Histopathology is observed in the Glandular stomach.

Hypertrophy parietal cells was present in all 1,2-Dichlorobenzene treated groups in a dose dependent manner and up to slight degree.

Overall it can be concluded: in the MNT (Charles River, 2017) 1,2-Dichlorobenzene is not clastogenic or aneugenic in the bone marrow micronucleus test of male rats up to a dose of 500 mg/kg (the maximum tolerated dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.

In the comet assay no genotoxic effect was observed in bone marrow, duodenum and liver. In stomach, the result of the Comet assay was equivocal and additional investigation is required (Charles River, 2017).

Based on the equivocal results in the Comet assay in the stomach we do propose the following follow-up activities:

Re-conduct a Comet assay in glandular stomach and duodenum in the same strain of rats (Wistar) in another laboratory with more experience and tighter historical control data not as broad as the initial laboratory. In addition, histopathology will be included right from the beginning in the study protocol to set observations into perspective for this irritating compound. The study is scheduled for February 2018 and the audited draft will be available end of March. Based on these results potential tier 2 follow up activities will be considered.