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EC number: 200-864-0 | CAS number: 75-35-4
The time-weighted mean actual concentrations are shown in the table below.
Time-weighted mean actual concentration (mg/L)
3.7 +/- 0.2
4.3 +/- 0.3
8.0 +/- 0.7
6.5 +/- 0.4
9.1 +/- 0.5
17.1 +/- 0.8
18.4 +/- 0.7
20.5 +/- 0.9
38.9 +/- 1.1
38.7 +/- 1.8
38.1 +/- 1.8
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.
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.
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.
TEST ATMOSPHERE CHARACTERIZATION
27.2 +/- 1.2
25.1 +/- 1.5
25.9 +/- 5.3
3.3 +/- 0.1
3.0 +/- 0.2
1.1 +/- 0.03
0.12 +/- 0.003
0.116 +/- 0.007
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
Bone Marrow (%)
200 mg/Kg (EMS)
RESULTS OF THE COMET ASSAY - TAIL INTENSITY
Overall tail intensity (%) and standard deviation (SD) per dose and organ are tabulated here below:
1,1-dichloroethylene 25 mg/L
EMS 200 mg/kg
1,1-dichloroethylene 3 mg/L
1,1-dichloroethylene 1 mg/L
1,1-dichloroethylene 0.1 mg/L
*p<0.05 (Students t test)
**p<0.01 (Students t test)
*** p<0.001 (Students t test)
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.
Relevant DNA damage
No test substance related findings
No relevant DNA damage
Severe findings (tubular degeneration)
DNA damage questionable
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.
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