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Toxicological information

Genetic toxicity: in vivo

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Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: 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:
other: Recent study according to OECD and GLP guidelines.
Cross-reference
Reason / purpose:
reference to other study
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: It concerns a maximum tolerated dose study which was performed prior to a Comet Assay. The test procedures are in line with OECD 403 (acute toxicity testing) and OECD 489 (Comet assay).
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
other: OECD 403 (acute toxicity testing)
Qualifier:
equivalent or similar to
Guideline:
other: OECD 489 (Comet Assay)
Principles of method if other than guideline:
Groups of three male and three female Wistar Han rats were exposed (nose only) by inhalation for three consecutive days during four hours each day to a target concentration of 4 mg/L (1,000 ppm), 8 mg/L (2,000 ppm), 19 mg/L (4,750 ppm), 40 mg/L (10,000 ppm) and 80 mg/L (20,000 ppm). Clinical signs and body weights were observed once daily. Macroscopic examination was performed on Day 3, lung, liver and kidney were weighted and preserved, the cells of the bronchial alveolar lavage fluid of the lungs were counted and differentiated and blood was collected for clinical biochemistry.

The test is not performed in accordance with an OECD guideline for repeated dose testing via inhalation, but the study procedures described in this protocol are based on the following guidelines:
- Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Section 4, Health Effects. No.403, "Acute Inhalation Toxicity", September 2009.
- ICH M3 (R2): Note for guidance on non-clinical safety studies for the conduct of human clinical trials and marketing authorization for pharmaceuticals, December 2009.
- ICH S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use; Draft ICH Consensus Guideline, Step 4 of the Process, November 2011
- OECD 489: In vivo mammalian alkaline Comet Assay, September 2014
- Tice, R. R., Agurell, E., Anderson, D., Burlinson, B., Hartmann, A., Kobayashi, H., Miyamae, Y., Rojas, E., Ryu, J.-C. and Sasaki, Y.F. (2000). Single Cell Gel/Comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ mol mutagen 35, p 206 - 221.
- Smith CC, Adkins DJ, Martin EA, O'Donovan MR. (2008). Recommendations for design of the rat comet assay. Mutagenesis 23(3), p 233 – 240
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Species: Rat, Wistar strain: Crl:WI(Han) (outbred, SPF-Quality).
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 8-12 weeks
- Weight at study initiation: Animals used within the study were of approx. the same age and body weight variation that did not exceed +/- 20% of the sex mean.
- Housing: Group housing of maximally 3 animals per sex per cage in labelled Makrolon cages (type IV; height 18 cm.) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom).
- Identification: tattoo on hind leg.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF Spezialdiäten GmbH, Soest, Germany) except during exposure to the test substance.
- Water (e.g. ad libitum): Free access to tap water except during exposure to the test substance.
- Acclimation period: at least 5 days before the start of treatment under laboratory conditions.
- Health inspection: at least prior to exposure. It was ensured that the animals were healthy and without any abnormality that might affect the study integrity.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24°C
- Humidity (%): 40-70%
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12-hrs light / 12-hrs dark cycle
Route of administration:
inhalation: vapour
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
TYPE OF INHALATION EXPOSURE: nose only

EXPOSURE CHAMBER
Animals were exposed to the test substance via the inhalation route. For this purpose the animals were placed in restraining tubes, which were connected to the exposure chamber.
The design of the exposure chamber was based on the flow past nose-only inhalation chamber (Am. Ind. Hyg Assoc. J. 44(12): 923-928, 1983). The chamber consisted of animal sections with eight animal ports each. Each animal port had its own test atmosphere inlet and exhaust outlet. The number of animal sections and number of open inlets was adapted to the air flow in such a way that at each animal port the theoretical air flow was at least 1 L/min. The main inlet of the test atmosphere was located at the top section and the main outlet was located at the bottom section. The direction of the flow of the test atmosphere guaranteed a freshly generated atmosphere for each individual animal. All components of the exposure chamber in contact with the test substance were made of stainless steel, glass, rubber or plastic. To avoid exposure of the personnel and contamination of the laboratory the exposure chamber was placed in a fume hood.

GENERATION OF TEST ATMOSPHERE
The test substance was put on ice in order to maintain a constant temperature. A vapor was generated by passing a stream of pressurized air through the test substance. The primary vapor was diluted with pressurized air in order to obtain the desired concentration and was subsequently passed through the exposure chamber.
From the exposure chamber the test atmosphere was passed through a filter before it was released to the exhaust of the fume hood.

STUDY DESIGN
The study was performed following a stepwise exposure scenario. Based on available toxicity data, the study was started with the exposure of three males and three females at a starting target concentration of 4 mg/L (1,000 ppm). Based on the results, additional groups of three males and three females were exposed to target concentrations of 8 mg/L (2,000 ppm), 20 mg/L (5,000 ppm), 40 mg/L (10,000 ppm) and 80 mg/L (20,000 ppm). The groups were exposed for three consecutive days and the exposure time was 4 hours each day.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
TEST ATMOSPHERE CHARACTERIZATION

Nominal concentration
The nominal concentration was calculated by dividing the amount of test substance used by the volume of pressurized air (average air flow times exposure time) entering the exposure chamber used for exposure of the animals. Due to the small volume of the exposure chamber the equilibrium time was negligible. The volume of air was calculated from the average air flow (measured by means of thermal mass flow meters and was recorded regularly, preferably in 30 minute intervals) and the exposure time.

Actual concentration
The actual concentration was determined at least five times during each exposure period each day. Sample intervals were appropriately spaced over the exposure periods and additional samples were taken in case the air flow was readjusted. Samples were drawn for at least 1 minute with a sample flow of 2 L/minute from the test atmosphere through a tube mounted in one of the free animal ports of the middle section of the exposure chamber. Samples were collected in glass collector bulbs which had a volume between 125 and 500 mL. If needed the samples were diluted with pressurized air and transferred into Tedlar gas bags. The volume of diluting air was measured by means of a dry gas meter (type G 1.6, Actaris Meterfabriek B.V., Dordrecht, The Netherlands). The samples were transferred to the analytical department for immediate analyses. Analyses of the test atmosphere was done according to a validated Gas Chromatographic method (method developed under WIL Research Europe project: 509161). The mean concentration(s) and standard deviation(s) were calculated for each exposure day.

ANALYTICAL METHOD
- Instrument: Gaschromatograph 6890N (Agilent Technologies, Palo Alto, CA, USA)
- Injector: CombiPal (CTC analytics, Zwingen, Switzerland)
- Detector: Flame ionisation detector (Agilent Technologies)
- Column: DB-1; 30 m x 320 μm i.d., df = 5 μm (J&W Scientific, Folsom, CA, USA)
- Carrier gas: helium
- Carrier gas flow: 2 ml/min
- Injection: Programmed temperature vaporizer (PTV) Spilt
- PTV Mode: Split
- Split ratio: 10:1
- Split flow: 20 ml/min
- Gas saver: Off
- Injection volume: 25 μl (manual injection)
- PTV temperature program: Temperature: 100°C
- Oven temperature program: Temperature: 100°C
- FID detection temperature: 300°C
- Hydrogen: 40 ml/min
- Air: 450 ml/min
- Nitrogen (make-up): 50 ml/min
Duration of treatment / exposure:
4h/d
Frequency of treatment:
3 consecutive days
Remarks:
Doses / Concentrations:
4 mg/L
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
8 mg/L
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
19 mg/L
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
40 mg/L
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
80 mg/L
Basis:
nominal conc.
No. of animals per sex per dose:
3 males and 3 females per dose
Control animals:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: Prior to dosing, on day 2 and on day 3 (termination)

CLINICAL CHEMISTRY: Yes
- Collection of blood from the retro-orbital sinus.
Blood samples were drawn from the retro-orbital sinus of all surviving animals into tubes Li-heparin (0.5 mL) for clinical biochemistry parameters (tubes: Greiner Bio-One GmbH, Kremsmünster, Austria).
- Paramaters: Alanine aminotransferase (ALAT), Aspartate aminotransferatse (ASAT), Total protein, Albumin, Total Bilirubin, Urea, Creatinine, Potassium
Sacrifice and pathology:
All animals were deeply anaesthetized using an intraperitoneal buphrenorphine / dexmedetomidine/ketamine injection after completion of the inhalation exposure on Day 3.

Macroscopy and weighing of the lungs, kidneys and liver.
All animals were subjected to necropsy and descriptions of all internal macroscopic abnormalities were recorded. Particular attention was given to any changes in the respiratory tract, lungs, liver and kidneys. The lungs, kidneys and liver were removed and weighed.

Bronchoalveolar lavage (BAL).
After weighing, the lungs were infused twice with 5 mL sterile 0.9 % saline (cold, i.e. taken from refrigerator) and the BAL fluid (BALF) was put on ice. Subsequently, the BALF was centrifuged (300 g, 10 min, 4°C) and the supernatant was removed. The cell pellet was resuspended in 1 mL of sterile saline.Aliquots of the cell suspensions were used to determine cell numbers and white blood cells (differentiated at least for neutrofils).

Fixation of the lungs, liver and kidneys.
The lungs, kidneys and liver were fixed with 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands). Lungs were fixed by infusion following the lavage. Identification marks were also fixed. No histotechnology or histopathology was performed.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality occurred at any of the exposure levels.
Clinical signs:
- At 4 mg/L, slow breathing was seen for the animals during exposure. After exposure, hunched posture and piloerection were seen for two females on Day 1 only.
- At 8 mg/L, slow breathing was seen for the animals during exposure. After exposure, lethargy, hunched posture and piloerection were seen for the animals on Day 1 only. One female showed hunched posture and piloerection on Day 2.
- At 19 mg/L, slow breathing was seen for the animals during exposure. After exposure, hunched posture and piloerection were seen for the animals on Day 1 only.
- At 40 mg/L, slow breathing was seen for the animals during exposure. After exposure, lethargy, hunched posture and piloerection were seen for the animals on Day 1 only.
- At 80 mg/L, lethargy, hunched posture, uncoordinated movements, labored respiration, ptosis and/or piloerection were seen for the animals on Days 1, 2 and or 3.

BODY WEIGHTS AND WEIGHT GAIN
Body weight loss with a maximum of 8% was seen for the animals exposed to 4 and 8 mg/L. Higher body weight loss exceeding 10% was seen for the animals exposed to 19, 40 and 80 mg/L.

ORGAN WEIGHTS
A small exposure related increase in lung: body weight ratio was seen. There were no evident exposure related changes for the liver and kidney ratios.

MACROSCOPIC FINDINGS
No abnormalities were found at macroscopic post mortem examination of the animals.

CLINICAL CHEMISTRY
Clinical biochemistry showed an increase in the liver and kidney parameters (Aspartate aminotransferase activity (ASAT), alanine aminotransferase activity (ALAT), total bilirubin, urea, creatinine) with the highest increases observed at 40 and 80 mg/L.

CELL DIFFERENTIATION BRONCHIAL ALVEOLAR FLUID
A marked increase in total cell count of the bronchial alveolar fluid was seen at 80 mg/L. The cell differentiation showed an increase in neutrophils and a reduction for lung macrophages. No relevant changes were seen for the lymphocytes. Some Eosinophils were seen over all exposure groups and monocytes were seen for the animals exposed to 40 mg/L.
Dose descriptor:
other: Maximum Tolerated Dose (MTD)
Effect level:
40 - 80 mg/L air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: clinical chemistry: increased liver and kidney parameters; cell differentiation bronchial alveolar fluid
Critical effects observed:
not specified

The time-weighted mean actual concentrations are shown in the table below.

 

Time-weighted mean actual concentration (mg/L)

Exposure group

Day 1

Day 2

Day 3

4 mg/L

(1000 ppm)

3.7 +/- 0.2

4.4 +/-0.2

4.3 +/- 0.3

8 mg/L

(2000 ppm)

8.0 +/- 0.7

6.5 +/- 0.4

9.1 +/- 0.5

19 mg/L

(4750 ppm)

17.1 +/- 0.8

18.4 +/- 0.7

20.5 +/- 0.9

40 mg/L

(10000 ppm)

38.9 +/- 1.1

38.7 +/- 1.8

38.1 +/- 1.8

80 mg/L

(20000 ppm)

79.2 +/3.7

83.8 +/- 2.6

79.6 +/- 2.7

The concentration measurements equally distributed over time showed that the substance concentrations were sufficiently stable. The variations were caused by adjustments to the generation equipment and were considered not to have affected the exposure level. By calculation the time- weighted concentrations the influence of these variations were taken into account.

Conclusions:
The increases for biochemical liver and kidney parameters and increases in lung cell counts and neutrophils do suggest adverse changes in lung, liver and kidney but these changes did not result in macroscopic abnormalities. Overall, the effects seen after exposure to 80 mg/L were considered toxicologically significant. Considering the limited number of animals and based on the data available, the MTD for 1,1- dichloroethylene after a 3-day nose only inhalation exposure was considered to be between 40 and 80 mg/L. There was no evidence suggesting a difference in sensitivity between the sexes.
Executive summary:

The range finding and Maximum Tolerated Dose (MTD) study with 1,1-dichloroethylene assesses adverse effects observed after a 3-day nose-only inhalation exposure in Wistar rats. This MTD study was performed in order to identify any difference in sex sensitivity and to establish the MTD to be used in the in vivo Comet test (WIL project 509160). The inhalation route was selected, as it is the relevant route in humans.

Groups of three male and three female Wistar Han rats were exposed (nose only) by inhalation for three consecutive days during four hours each day to1,1 -dichloroethylene (vapour). The study was performed following a stepwise exposure regime. The starting target concentration was of 4 mg/L (1,000 ppm). Based on the results at this concentration, additional groups of three males and three females were exposed to target concentrations of 8 mg/L (2,000 ppm), 19 mg/L (4,750 ppm), 40 mg/L (10,000 ppm) and 80 mg/L (20,000 ppm). Clinical signs and body weights were observed once daily. Macroscopic examination was performed on Day 3, lung, liver and kidney were weighted and preserved, the cells of the bronchial alveolar lavage fluid of the lungs were counted and differentiated and blood was collected for clinical biochemistry.

No mortality occurred at any of the exposure levels. Clinical signs observed included slow or labored breathing, lethargy, hunched posture, ptosis and piloerection, and were dose dependent. A body weight loss of max. 8% was seen for animals exposed to 4 and 8 mg/L. Higher body weight loss exceeding 10% was seen for animals exposed to 19, 40 and 80 mg/L. A small exposure related increase in lung/body weight ratio was seen, however no statistical analysis was performed in the study and therefore it is not possible to conclude in the relevance of this finding.

There was no evident exposure related change for the liver and kidney ratios.

No abnormalities were found at macroscopic post mortem examination of the animals.

Clinical biochemistry showed an increase in the liver and kidney parameters (ASAT, ALAT, total bilirubin, urea and creatinine) with the highest increases observed at 40 and 80 mg/L.

Cell differentiation bronchial alveolar fluid showed a marked increase in total cell count of the bronchial alveolar fluid was seen at 80 mg/L, , an increase in neutrophils and a reduction for lung macrophages. No relevant changes were seen for the lymphocytes. Some eosinophils were seen over all exposure groups and monocytes were seen for the animals exposed to 40 mg/L.

The increases for biochemical liver and kidney parameters and increases in lung cell counts and neutrophils do suggest adverse changes in lung, liver and kidney but these changes did not result in macroscopic abnormalities.

Overall, the effects seen after exposure to 80 mg/L were considered toxicologically significant.

Considering the limited number of animals and based on the data available, the MTD for 1,1- dichloroethylene after a 3-day nose only inhalation exposure was considered to be between 40 and 80 mg/L. There was no evidence suggesting a difference in sensitivity between the sexes.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD No. 489:In Vivo Mammalian Alkaline Comet Assay (adopted September 26, 2014)
Deviations:
yes
Remarks:
Deviations not affecting the integrity of the study.
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian comet assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Physical state: clear colourless liquid
- Analytical purity: > 99%
- Lot/batch No.: 5D235
- Storage condition of test material: in freezer (≤ -15°C) protected from light
- Stable under storage conditions until: 13 December 2015 (retest date)

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Species: Rat, Wistar strain: Crl:WI(Han) (outbred, SPF-Quality).
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 7-8 weeks
- Weight at study initiation: Animals used within the study were of approx. the same age and body weight variation that did not exceed +/- 20% of the sex mean.
- Fasting period before study: Positive control animals weredosed by oral gavage. These animals were deprived of food 2-3 hours prior to each dosing and until 2-3 hours after administration of the positive control substance.
- Housing: Group housing of maximally 5 animals per sex per cage in labelled Makrolon cages (type IV; height 18 cm.) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom).
- Identification: tail mark with indelible ink.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF Spezialdiäten GmbH, Soest, Germany) except during exposure to the test substance.
- Water (e.g. ad libitum): Free access to tap water except during exposure to the test substance.
- Acclimation period: at least 5 days before the start of treatment under laboratory conditions.
- Health inspection: at least prior to exposure. It was ensured that the animals were healthy and without any abnormality that might affect the study integrity.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24°C
- Humidity (%): 40-70%
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12-hrs light / 12-hrs dark cycle

Administration / exposure

Route of administration:
inhalation: vapour
Vehicle:
- Vehicle(s)/solvent(s) used: pressurized air.
- Concentration of test material in vehicle: 0.1, 1, 3 and 25 mg/L.
Details on exposure:
TYPE OF INHALATION EXPOSURE: nose only

EXPOSURE CHAMBER
Animals were placed in restraining tubes, which were connected to the exposure chamber with a volume of approximately 150 mL.
The design of the exposure chamber is based on the flow past nose-only inhalation chamber (Am. Ind. Hyg Assoc. J. 44(12): 923-928, 1983). The chamber consisted of animal sections with eight animal ports each. Each animal port had its own test atmosphere inlet and exhaust outlet. The number of animal sections and number of open inlets were adapted to the air flow in such a way that at each animal port the theoretical air flow was at least 1 L/min. The main inlet of the test atmosphere was located at the top section and the main outlet at the bottom section. The direction of the flow of the test atmosphere guarantees a freshly generated atmosphere for each individual animal. All components of the exposure chamber in contact with the test substance were made of stainless steel, glass, rubber or plastic. To avoid exposure of the personnel and contamination of the laboratory the exposure chamber was placed in a fume hood.

GENERATION OF TEST ATMOSPHERE
The test substance was put on ice in order to maintain a constant temperature. A vapor was generated by passing a stream of pressurized air through the test substance. The primary vapor was diluted with pressurized air in order to obtain the desired concentration and was subsequently passed through the exposure chamber.
From the exposure chamber the test atmosphere was passed through a filter before it was released to the exhaust of the fume hood.

STUDY DESIGN
The study was performed following a stepwise exposure scenario. The target concentrations and sex(es) to be used were based on a MTD study (WIL project 509159). The MTD study showed no substantial differences between sexes in toxicity and the MTD was found to be between 40 and 80 mg/L based on clinical observations and body weight.

The main study was started with the inhalation exposure of five males to a target concentration of 25 mg/L for 4 hours during three consecutive day. This dose was initially considered (based on the Maximum Tolerated Dose study results) as an intermediate dose. Since significant DNA damage and histopathological findings were observed at that exposure level, the next group of five males was exposed to 3 mg/L in order to decrease the histopathological findings. Based on these results, the next group was exposed to 1 mg/L in order to further reduce the histopathological findings. In order to obtain information about
the dose response and to have an exposure level without adverse histopathological findings in any organ, the last group of five males was exposed to 0.1 mg/L.

TEST ATMOSPHERE CHARACTERIZATION
Nominal concentration
The nominal concentration was calculated by dividing the amount of test substance used by the volume of pressurized air (average air flow times exposure time) entering the exposure chamber used for exposure of the animals. Due to the small volume of the exposure chamber (approximately 150 mL) the equilibrium time was negligible. The volume of air was calculated from the average air flow (measured by means of thermal mass flow meters and was recorded regularly, preferably in 30 minute intervals) and the exposure time.

Actual concentration
The actual concentration was determined at least ten times during each exposure period each day. Sample intervals were appropriately spaced over the exposure periods and additional samples were taken in case the air flow was readjusted. Samples were drawn for at least 1 minute with a sample flow of 2 L/minute from the test atmosphere through a tube mounted in one of the free animal ports of the middle section of the exposure chamber. Samples were collected in glass collector bulbs which had a volume between 125 and 500 mL. If needed the samples were diluted with pressurized air and transferred into Tedlar gas bags. The volume of diluting air was measured by means of a dry gas meter (type G 1.6, Actaris Meterfabriek B.V., Dordrecht, The Netherlands).
The samples were transferred to the analytical department for immediate analyses. Analyses of the test atmosphere was done according to a validated Gas Chromatographic method (method developed under WIL Research Europe project: 509161). The mean concentration(s) and standard deviation(s) were calculated for each exposure day.
Duration of treatment / exposure:
4 hours/day
Frequency of treatment:
3 consecutive days
Post exposure period:
None, animals were sacrifized 4 hours after completion of the last inhalation exposure, or within 2-6 hours after the last oral dosing of the positive control substance.
Doses / concentrationsopen allclose all
Dose / conc.:
25 mg/L air (nominal)
Remarks:
Corresponds to 6350 ppm; 25000 mg/m3
Dose / conc.:
3 mg/L air (nominal)
Remarks:
Corresponds to 750 ppm; 3000 mg/m3)
Dose / conc.:
1 mg/L air (nominal)
Remarks:
Corresponds to 250 ppm; 1000 mg/m3)
Dose / conc.:
0.1 mg/L air (nominal)
Remarks:
Corresponds to 25 ppm; 100 mg/m3)
No. of animals per sex per dose:
5 males per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
POSITIVE CONTROL
- Positive control substance: ethylmethanesulphonate (EMS)
- Route of administration: oral gavage
- Doses / concentrations: 200 mg/kg bw

TREATMENT OF POSITIVE CONTROL ANIMALS BY ORAL GAVAGE
- Method: Oral gavage, using plastic feeding tubes. The positive control formulation was stirred on a magnetic stirrer during dosing.
- Rationale: The oral route and frequency was selected based on previous studies and laboratories historical data base. The oral route was considered suitable since both routes, inhalation and oral, provides systemic availability of the test substance.
- Fasting: Animals were deprived of food 2-3 hours prior to each dosing and until 2- 3 hours after administration of the positive control substance. Water was available.
- Frequency: Animals were dosed twice; between 16-26 and 2-6 hours prior to removal of the tissues for the Comet Assay.
- Dose volume: 10 mL/kg bw for each dose.
- Dose level: 200 mg/kg bw.

Examinations

Tissues and cell types examined:
Lung, liver, kidney and bone marrow.
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Tissues for the Comet Assay were collected once within 4 hours after completion of the last inhalation exposure, and within 2 and 6 hours after the last oral dosing of the positive control substance. All animals were deeply anaesthetized using a buphrenorphine / dexmedetomidine / ketamine injection prior to necropsy.

Lung, liver and kidney tissue.
A small part was dissected from the lung, liver and kidney and put in labelled containers with cold medium for the examination on DNA damage with the alkaline Comet Assay. The remaining part of the organs were fixated in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands) for histopathological examination.

The isolation method of cells for the Comet assay was based on the publication of Hu et al (2002). A portion of 0.4-0.7 gram from the lung, liver or kidney was removed and minced thoroughly on aluminium foil in ice. The minced tissue was added to 10 mL of collagenase (20 Units/mL; Sigma Aldrich, Zwijndrecht, The Netherlands) dissolved in HBSS (Ca2+- and Mg2+-free; Life technologies, Breda, The Netherlands) and incubated in
a shaking waterbath at 37 °C for 20 minutes. Thereafter, a low centrifugation force was applied to remove large undigested liver debris (40 g for 5 min). The supernatant was collected and centrifuged to precipitate the cells (359 g for 10 min). The supernatant was removed and the cell pellet was resuspended in ice cold HBSS (Ca2+- and Mg2+-free) and kept on ice.

Bone marrow.
One femur containing the bone marrow was dissected and the bone marrow was transferred to labelled containers with cold medium for the examination on DNA damage with the alkaline Comet Assay. From the other femur, bone marrow smears were prepared. No further examination on the smears were performed within this study.

To isolate cells for the Comet assay, the femur was freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal becoma visible. The bone was flushed with approximately 4 mL of ice cold HBSS (HBSS; Ca2+- and Mg2+-free). The cell suspension was collected and stored on ice. A low centrifugation force was applied to remove cell debris (40 g for 5 min). Thereafter the supernatant was centrifuged for 5 min at 359 g. The supernatant was removed and the cell pellet was resuspended in ice cold HBSS (Ca2+- and Mg2+-free) and kept on ice.

DETAILS OF SLIDE PREPARATION:

To 20 μL of the cell suspension, 280 μL melted low melting point agarose (LMAgarose; Trevigen, Gaithersburg, USA) was added. The cells were mixed with the LMAgarose and 50-60 μL was layered on a precoated Comet slide (Trevigen) in duplicate. Three slides per tissue were prepared. The slides were marked with the study identification number, animal number and group number. The slides were incubated for at least 10-15 minutes in the refrigerator in the dark until a clear ring appears at the edge of the Comet slide area.

The cells on the slides were overnight immersed in prechilled lysis solution (Trevigen) in the refrigerator. After this period the slides were immersed/rinsed in neutralization buffer (0.4 M Tris-HCl pH 7.4; Sigma-Aldrich) for approximately 5 min. The slides were then placed in freshly prepared alkaline solution for 30-55 minutes at room temperature in the dark. The slides were placed in the electrophoresis unit just beneath the alkaline buffer solution and the voltage was set to 1 Volt/cm. The
electrophoresis was performed for 30 minutes under constant cooling (actual temperature 4.5 –7.0°C). After electrophoresis the slides were immersed/rinsed in neutralization buffer for 5 minutes.
The slides were subsequently immersed approximately 5 minutes in absolut ethanol (≥99.6%; Merck, Darmstadt, Germany) and allowed to dry at room temperature. The slides were stained for approximately 5 minutes with the fluorescent dye SYBR® Gold (Life Technologies, Breda, The Netherlands) in the refrigerator. Thereafter the slides were washed with Milli-Q water and allowed to dry at room temperature in the dark.

METHOD OF ANALYSIS:

To prevent bias during Comet scoring, slides were randomly coded (per tissue) before examination of the Comets. An adhesive label with study identification number and code were placed over the marked slide. 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 (protocol deviation 1). The goal was to
score 50 comets per agarose circle. In case it was not possible to score 50 comets per agarose circle the remaining number of comets was scored on an additional agarose circle.
The following criteria for scoring of Comets were used:
- Only horizontal orientated Comets were scored, with the head on the left and the tail on the right.
- Cells that showed overlap or were not sharp were not scored.

OTHER:

Determination of cell viability
The viability of the cells after isolation was determined by manually counting the number of viable cells using trypane blue staining. Due to the fact that it is important to work quickly when preparing the slides the viability was only checked for 1 animal per group.

Lysis, electrophoresis and staining of the slides
The cells on the slides were overnight immersed in prechilled lysis solution (Trevigen) in the refrigerator. After this period the slides were immersed/rinsed in neutralization buffer (0.4 M Tris-HCl pH 7.4; Sigma-Aldrich) for approximately 5 min. The slides were then placed in freshly prepared alkaline solution for 30-55 minutes at room temperature in the dark. The slides were placed in the electrophoresis unit just beneath the alkaline buffer solution and the voltage was set to 1 Volt/cm. The
electrophoresis was performed for 30 minutes under constant cooling (actual temperature 4.5 – 7.0°C). After electrophoresis the slides were immersed/rinsed in neutralization buffer for 5 minutes.
The slides were subsequently immersed approximately 5 minutes in absolut ethanol (≥99.6%; Merck, Darmstadt, Germany) and allowed to dry at room temperature. The slides were stained for approximately 5 minutes with the fluorescent dye SYBR® Gold (Life Technologies, Breda, The Netherlands) in the refrigerator. Thereafter the slides were washed with Milli-Q water and allowed to dry at room temperature in the dark.

Evaluation criteria:
ACCEPTABILITY OF THE ASSAY
The in vivo comet assay 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 increase in the percentage Tail Intensity compared to the negative control treated animals. The positive control data was analyzed by the Students t test (one-sided, p < 0.05)

EVALUATION CRITERIA
A test compound is considered positive in the Comet assay (in a tissue) if the following criteria are met: It induces a biologically as well as a statistically significant (Students t test, one-sided, p < 0.05) increase in percentage Tail Intensity at one or more of the tested concentrations.

A test compound is considered as negative in the Comet assay (in a tissue) if the following criteria are met: None of the tested concentrations show a statistically (Students t test, one-sided, p < 0.05) or biological significant dose-dependent increase in percentage Tail Intensity.
Statistics:
ToxRat Professional v3.0.0 was used for statistical analysis of the data.

Results and discussion

Test resultsopen allclose all
Sex:
male
Genotoxicity:
positive
Remarks:
Statistical significant and biological relevant DNA damage without adverse histopathological findings was observed in lung cells (at 1 mg/L) and kidney cells (at 3, 1, 0.1 mg/L).
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Sex:
male
Genotoxicity:
ambiguous
Remarks:
Biologically relevant increase in DNA damage seen after exposure to 25 mg/L (in lung, liver, kidney), 3 mg/L (in lung, liver) and 1 mg/L (in liver) might have been caused by a genotoxic effect and/or histopathologic effect.
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
See cross-referenced study.

RESULTS OF DEFINITIVE STUDY - OBSERVATIONS
- Mortality: No mortality occurred in any of the animals.
- Clinical signs: At 25 and 1 mg/L, hunched posture and piloerection were seen for all animals on Day 1 only. No clinical signs were seen for the animals exposed to 3 and 0.1 mg/L, for the negative control animals exposed to air and the orally dosed positive control animals.
- Body weights: At 25 mg/L exposure level, body weight loss between 12 and 16% was seen for the animals compared to Day 1 values. At 3 and 1 mg/L exposure level, some slight to moderate body weight loss between 1 and 3% was seen. At 0.1 mg/L exposure level, the body weight gain of the animals was within the range as seen for the negative control animals.
- Macroscopic findings: Test substance related macroscopic findings consisted of reddish discoloration of the lungs and dark red discoloration of the liver found for one experimental animal exposed to 25 mg/L and red brown discoloration of the kidneys was found for one animal exposed to 3 mg/L. No other abnormalities were found at macroscopic post mortem examination of the experimental or negative control animals. Reddish foci on the thymes were found for one positive control animal.

RESULTS OF DEFINITIVE STUDY - VIABILITY
The viability of one single cell suspension per tissue per group was assessed by using trypan blue.
The viability of the single suspensions was 94%-100% for kidney, 98%-100% for liver, 97%-100% for lung and 84%-100% for bone marrow. For details, see table.

RESULTS OF DEFINITIVE STUDY - KIDNEYS
Histopathology
- 25 mg/L: Tubular degeneration, mainly proximal tubules was present in 5/5 males (2 minimal, 2 slight, 1 moderate)

Comet
A statistically significant increase (Students t test p<0.05) of the Tail Intensity (%) was observed after treatment with 1,1-dichloroethylene at all test concentrations. DNA damage expressed as percentage Tail Intensity increased from 4.37% to 68.72%, 11.57% to 57.25%, 26.58% to 35.59% and 13.75% to 54.95% at 25, 3, 1 and 0.1 mg/L, respectively.The Tail intensities (%) after treatment at these four concentraties were clearly above the historical control data range (2.31% – 24.02%) and the increases are therefore considered biologically relevant. More details by experiment in table below.

RESULTS OF DEFINITIVE STUDY - LIVER
Histopathology
- 25 mg/L: Single cell necrosis, centrilobular/bridging was present in 5/5 males (1 minimal, 1 slight, 2 moderate, 1 marked).
- 3 mg/L: Single cell necrosis, centrilobular/bridging was present in 5/5 males (4 slight, 1 moderate); Hepatocellular hypertrophy, centrilobular was present in 5/5 males (1 minimal, 4 slight).
- 1 mg/L: Single cell necrosis, centrilobular/bridging was present in 5/5 males (1 slight, 3 moderate, 1 marked); Hepatocellular hypertrophy, centrilobular was present in 5/5 males (3 slight, 2 moderate).
- 0.1 mg/L: Hepatocellular hypertrophy, centrilobular was present in 5/5 males (5 minimal); Cytoplasmic alteration centrilobular was present in 5/5 males (5 slight). The histopathological findings observed in the liver at 0.1 mg/L were, in the absence of hepatocellular necrosis, considered indicative of enzyme induction and were considered test substance related effects but not adverse.

Comet
A statistically significant increase (Students t test p<0.05) of the Tail Intensity (%) was observed after treatment with 1,1-dichloroethylene at 25, 3 and 1 mg/L. DNA damage expressed as percentage Tail Intensity increased from 3.65% to 19.96%, 15.38% to 33.29%, 16.35% to 44.39 at 25, 3, and 1 mg/L, respectively. The Tail intensities (%) after treatment at 3 and 1 mg/L were above the historical control data range (0.13% – 21.13%) and the increases are therefore considered biologically relevant. The percentage DNA damage observed at the highest test concentration of 25 mg/L was with 19.96% just within the historical control data range (0.13% – 21.13%). Although just within the historical control data range the induction was considered biological relevant because of a significant 5.5-fold induction. At the lowest test concentration of 0.1 mg/L no statistically significant increase of DNA damage was observed. More details by experiment in table below.

RESULTS OF DEFINITIVE STUDY - LUNGS
Histopathology
- 25 mg/L: Degeneration / regeneration bronchiolar epithelium was present in 5/5 males (1 slight, 4 moderate); Lymphoid depletion BALT was present in 5/5 males (3 slight, 2 moderate); Inflammatory cell infiltrate peribronchial was present in 5/5 males (2 slight, 3 moderate).
- 3 mg/L: Regeneration bronchiolar epithelium was present in 5/5 males (4 minimal, 1 slight); Inflammatory cell infiltrate peribronchial was present in 4/5 males (4 minimal).

Comet
A statistically significant increase (Students t test p<0.05) of the Tail Intensity (%) was observed after treatment with 1,1-dichloroethylene at 25, 3 and 1 mg/L. DNA damage expressed as percentage Tail Intensity increased from 4.61% to 32.04%, 19.72% to 50.04%, 15.05% to 81.64 at 25, 3, and 1 mg/L, respectively. The Tail intensities (%) after treatment at 25, 3 and 1 mg/L were above the historical control data range (0.69% – 18.05%) and the increases are therefore considered biologically relevant. At the lowest test concentration of 0.1 mg/L no statistically significant increase of DNA damage was observed. More details by experiment in table below.

RESULTS OF DEFINITIVE STUDY - BONE MARROW
Histopathology
Not applicable.

Comet
No statistically significant increase in the mean Tail Intensity (%) was observed in bone marrow cells of 1,1-dichloroethylene-treated male animals compared to the negative control treated animals at 25, 1 and 0.1 mg/L. At a dose of 3 mg/L, DNA damage expressed as percentage Tail Intensity increased 2.2-fold from 5.31% in the negative controls to 11.64% (statistically significant induction; Students t test p<0.05) in 1,1-dichloroethylene-treated animals. The DNA damage at 3 mg/L was within the historical control data range of 1.92% – 17.26% and therefore the biological relevance of the increase is questionable. More details by experiment in table below.

ACCEPTABILITY OF THE ASSAY

The Comet Assay showed that all experiments passed the acceptance criteria:

- The negative control Tail Intensities in kidney, liver, lung and bone marrow were within the
 historical control data range in all experiments with exception of kidney experiment 3 (1 mg/L) were the mean value was with 26.58% just above the upper limit of the range (24.02%) and lung experiment 2 (3 mg/L) were the mean value was with 19.72% just above the upper limit of the range (18.05%). Since the values were only just above the upper range of the historical data and the positive control caused severe DNA damage with a Tail Intensity of 92.93% and 93.42% in kidney experiment 3 (1 mg/L) and lung experiment 2 (3 mg/L), respectively, the sensitivity of these assays was considered not to be affected and both experiments were considered acceptable.


- EMS, the positive control item, induced a statistically significant increase in all experiments in all tissues.

Any other information on results incl. tables

TEST ATMOSPHERE CHARACTERIZATION

Exposure group

Time-weighted mean actual concentration (mg/L)

Experiment 1

Day 1

Day 2

Day 3

25 mg/L

27.2 +/- 1.2

25.1 +/- 1.5

25.9 +/- 5.3

0 mg/L

0

0

0

Experiment 2

Day 1

Day 2

Day 3

3 mg/L

3.3 +/- 0.1

3.0 +/- 0.2

3.3 +/- 0.1

0 mg/L

0

0

0

Experiment 3

Day 1

Day 2

Day 3

1 mg/L

1.1 +/- 0.03

1.1 +/- 0.03

1.1 +/- 0.03

0 mg/L

0

0

0

Experiment 4

Day 1

Day 2

Day 3

0.1 mg/L

0.12 +/- 0.003

0.12 +/- 0.003

0.116 +/- 0.007

0 mg/L

0

0

0

The actual concentrations were slightly above the target concentrations (up to 10% at 25, 3 and 1 mg/L and up to 20% at 0.1 mg/L). The concentration measurements equally distributed over each exposure period showed that the substance concentrations were sufficiently stable. The variations were caused by adjustments to the generation equipment and were considered not to have affected the exposure level. By calculation of the time weighted mean concentrations, the influence of these variations were taken into account for the determination of the overall exposure level.

The mean total airflow used for the concurrent control groups were lower than those used for the experimental groups exposed to 3, 1 and 0.1 mg/L exposure. However, the number of open inlets were adapted to the air flow in such a way that at each animal port the theoretical air flow was at least approximately 1 L/min so that for both groups the individual animals were comparably exposed.

RESULTS OF THE COMET ASSAY - VIABILITY OF CELL SUSPENSIONS

 Exposure group

Kidney (%)

 Liver (%)

 Lung (%)

 Bone Marrow (%)

 0 (Control)

25 mg/L

200 mg/Kg (EMS)

98

99

96 

100

99

100 

100

100

100 

98

96

99 

0 (Control)

3 mg/L

200 mg/Kg (EMS)

95

94

99 

100

100

98 

100

99

100 

95

97

92 

0 (Control)

1 mg/L

200 mg/Kg (EMS)

100

97

100 

100

98

100 

100

97

98 

96

98

98 

0 (Control)

0.1 mg/L

200 mg/Kg (EMS)

100

100

100 

100

100

100 

100

100

100 

100

100

84 

RESULTS OF THE COMET ASSAY - TAIL INTENSITY

Overall tail intensity (%) and standard deviation (SD) per dose and organ are tabulated here below:

 Exposure group  Kidney tail intensity % + (SD)   Liver tail intensity % + (SD)   Lungs tail intensity % + (SD)   Bone Marrow tail intensity % + (SD)

Experiment 1

Negative Control

1,1-dichloroethylene 25 mg/L

EMS 200 mg/kg

 

4.37 (0.76)

68.72*** (13.85)

97.05*** (1.55)

 

3.65 (3.34)

19.96*** (7.94)

92.71*** (4.80)

 

4.61 (0.69)

32.04* (24.40)

86.99*** (12.24)

4.32 (2.08)

4.00 (0.95)

80.27*** (10.03)

Experiment 2

Negative Control

1,1-dichloroethylene 3 mg/L

EMS 200 mg/kg

 

11.57 (2.56)

57.25***  (16.03)

96.62*** (1.89)

 

15.38 (4.30)

33.29*** (8.93)

99.04*** (0.21)

  

19.72 (9.75)

50.04*** (7.39)

93.42*** (1.80)

5.31 (1.30)

11.64** (3.18)

85.40***(2.81)

Experiment 3

Negative Control

1,1-dichloroethylene 1 mg/L

EMS 200 mg/kg

 

26.58 (5.07)

35.59* (7.90)

92.93*** (1.09)

 

16.35 (14.71)

44.39* (16.94)

94.53*** (1.62)

15.05 (7.08)

81.64*** (3.31)

90.60*** (2.81)

10.23 (1.99)

11.88 (4.42)

82.22*** (2.56)

Experiment 4

Negative Control

1,1-dichloroethylene 0.1 mg/L

EMS 200 mg/kg

 

13.75 (3.52)

54.95*** (11.02)

87.24*** (3.24)

 

14.45 (5.50)

16.87 (4.08)

86.75*** (2.86)

4.73 (1.01)

5.65 (1.38)

82.57*** (3.43) 

10.07 (3.02)

12.80 (7.10)

78.24*** (3.16)

*p<0.05 (Students t test)

**p<0.01 (Students t test)

*** p<0.001 (Students t test)

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): positive
It was concluded that 1,1-dichloroethylene should be considered as genotoxic after a 3-day inhalation (nose only) exposure of male Wistar rats.

Statistical significant and biological relevant DNA damage without adverse histopathological findings was observed in lung cells (at 1 mg/L) and kidney cells (at 3, 1, 0.1 mg/L) indicating that 1,1- dichloroethylene induced DNA damage in these cells. Therefore the increased DNA damage observed at these conditions is caused by a genotoxic effect.

Taking into account the histopathological adverse findings and possible indirect effects on the DNA damage, no conclusion could be drawn regarding the biologically relevant increase in DNA damage seen after exposure to 25 mg/L (in lung, liver, kidney), 3 mg/L (in lung, liver) and 1 mg/L (in liver). DNA damage at these conditions might have been caused by a genotoxic effect and/or pathologic effect.
Executive summary:

Groups of five Wistar male rats were exposed by (nose only) inhalation to a vapor of 1,1-dichloroethylene at target concentrations of 25, 3, 1 and 0.1 mg/L (6350, 750, 250 and 25 ppm or 25000, 3000, 1000 and 100 mg/m3, respectively) per group.

Concentrations and sex of rats were selected based on the results of a preliminary range-finding study (Maximum tolerated dose study by van Huygevoort, 2016).

Animals were exposed for 4 hours on three consecutive days using a stepwise exposure scenario. The fist concentration tested was 25 mg/L as it was considered (based on the MTD results) as an intermediate dose. Since significant DNA damage and histopathological findings were observed at that exposure level, the next group was exposed to 3 mg/L in order to decrease the histopathological findings. Based on these results, a third group of five male rats was exposed to 1 mg/L to further reduce the histopathological findings. Finally a fourth group of animals was dosed at 0.1 mg/L to obtain inforamtion about the dose response and to have an exposure level without adverse histopathological findings in any organs.

For each exposure session concurrent negative and positive control groups were used: five male animals were similarly exposed to pressurized air only (negative control) and five male animals were dosed orally with 200 mg/kg EMS for two consecutive days (positive controls)

For all animals, clinical signs were recorded once daily post-dose and body weights were determined once daily. Within 4 hours after the last inhalation exposure or within 2 and 6 hours after the last oral dosing, animals were subjected to macroscopic examination. Histopathology was performed on lung, liver and kidney.

Lung, liver, kidney and bone marrow were examined for DNA damage by using the alkaline in vivo Comet Assay (OECD guidance 489; adopted 26 September 2014). Cells from liver; lung and kideny were isolated and their viability determined manually by counting the number of viable cells using trypane blue staining. After the preparation of single cell suspensions from kidney, liver, lung and bone marrow, Comet slides were prepared for Comet scoring. ToxRat Professional v.3.0.0 was used for statistical analysis of the data.

The achieved exposure conditions (temperature and relative humidity) were considered appropriated for the relatively short exposure duration (4 hours). The concentration measurements were sufficiently for each exposure day.

Hunched posture and piloerection were observed on Day 1 only, in all animals dosed at 25 mg/L and 1 mg/L. No clinicals signs were observed in animals exposed to 3 and 0.1 mg/L and in animals exposed to air (negative controls). At 25 mg/L, body weight loss between 12 and 16% was seen for the animals compared to Day 1 values. At 3 mg/L and 1 mg/L exposure level, some slight to moderate body weight loss (between 1 and 3%) was observed. At 0.1 mg/L concentration level, the body weight of the animals was within the range of bosy weight of the negative controls.

Macroscopic findings reported consisted of reddish discoloration of the lungs and dark discoloration of the liver in one animal exposed to a dose of 25 mg/L and red brown discoloration of the kidneys in one animal exposed to a dose of 3 mg/L. No other abnormalities were observed in treated animals . No macroscopic findings were observed in negative control animals. Cilinical signs, body weights and macroscopic fidnigns for the positive control group were in the range of historical data.

The viability of the single suspensions was 94%-100% for kidney, 9¨%-100% for liver, 97%-100% for lung and 84%-100% for bone marrow.

The results of the histopathology and the Comet Assay on the tissues of the treated groups exposed to the test substance are summarized in the table below.

Organ

Dose (mg/L)

Histopathology

Comet Assay

Lung

25

Severe findings

Relevant DNA damage

 

3

Minimal findings

Relevant DNA damage

 

1

No test substance related findings

Relevant DNA damage

 

0.1

No test substance related findings

No relevant DNA damage

Liver

25

Severe findings

Relevant DNA damage

 

3

Severe findings

Relevant DNA damage

 

1

Severe findings

Relevant DNA damage

 

0.1

Non-adverse findings

No relevant DNA damage

Kidney

25

Severe findings (tubular degeneration)

Relevant DNA damage

 

3

No test substance related findings

Relevant DNA damage

 

1

No test substance related findings

Relevant DNA damage

 

0.1

No test substance related findings

Relevant DNA damage

Bone marrow

25

Not performed

No relevant DNA damage

 

3

Not performed

DNA damage questionable

 

1

Not performed

No relevant DNA damage

 

0.1

Not performed

No relevant DNA damage

Taking into account the histopathological adverse findings and possible indirect effects on the DNA damage, no conclusion could be drawn regarding the biologically relevant increase in DNA damage seen after exposure to 25 mg/L (in lung, liver, kidney), 3 mg/L (in lung, kidney) and 1 mg/L (in liver). DNA damage at these conditions might have been caused by a genotoxic effect and/or pathologic effect.

Statistical significant and biological relevant DNA damage without adverse histopathological findings was observed in lung cells (at 1 mg/L) and kidney cells (at 3, 1, 0.1 mg/L) indicating that 1,1- dichloroethylene induced DNA damage in these cells. Therefore the increased DNA damage observed at these conditions is caused by a genotoxic effect.

It was concluded that 1,1-dichloroethylene should be considered as genotoxic after a 3-day inhalation exposure of male Wistar rats.