Trisiloxane, 3-ethyl-1,1,1,3,5,5,5-heptamethyl-

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 March 2008 to 13 April 2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Deviations:

yes

Remarks:

refer to "Overall Remarks"

Principles of method if other than guideline:

Current guideline requirements require 150 cells to be scored/animal and that the historical control ranges presented should use C-charts, C-bar charts to identify the variability in the data set. This has not been undertaken.

GLP compliance:

yes

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

469-070-1

EC Name:

-

Cas Number:

17861-60-8

Molecular formula:

C9H26O2Si3

IUPAC Name:

4-ethyl-2,2,4,6,6-pentamethyl-3,5-dioxa-2,4,6-trisilaheptane

Test material form:

liquid

Details on test material:

Appearance: Clear colorless liquid

Specific details on test material used for the study:

Stability of test compound: Confirmed stable for the duration of the study

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Commonly used strain that has been used for many years

Sex:

male

Details on test animals or test system and environmental conditions:

Test animals:
Species: rat
Strain: Wistar
Age: 7-9 wks at dosing
Weight at dosing: 218-236g
Source: Harlan, Frederick
Acclimation period: 5 days
Diet: Harlan 2018C Certified Global Rodent Diet, ad libitum
Water: Municipal water, ad libitum
Housing: Five animals of the same sex/cage

Environmental conditions:
Temperature: 19-25°C
Humidity: 30-70%
Air changes: not stated
Photoperiod: 12h light/dark cycle

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Corn oil (dose volume 10 mL/kg bw)

Details on exposure:

Preparation of dosing solutions:
The test article was prepared and diluted in corn oil before treatment.

Duration of treatment / exposure:

Single oral dose

Frequency of treatment:

single

Post exposure period:

sampling of liver and stomach 4 and 24 h post a single oral dose

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 500 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Sacrifice time: 4 hours: 0, 1000, 15000, 2000 mg/kg bw, MMS: 50 mg/kg bw; No. of animals: 5/gp
Sacrifice time: 24 hours: 0, 1000, 1500, 2000 mg/kg bw; No. of animals: 5/gp

Control animals:

yes, concurrent vehicle

Positive control(s):

Methylmethanesulphonate (MMS) a widely used postive control for comet induction was used. A single oral dose, via gavage at 50 mg/kg bw (employing a dose volume of 10 mL/kg bw) was used

Examinations

Tissues and cell types examined:

Hepatocytes and stomach epithelial cells were isolated and a single cell preparation was prepared.

Details of tissue and slide preparation:

Details of tissue preparation:
- Liver:
The hepatocytes were isolated from the liver and the cell suspension was strained using a cell strainer and then kept on ice until slide preparation. The number of isolated living cells was determined by the trypan blue dye exclusion test.

- Stomach:
Stomachs were removed and cut open and washed free from food using mincing buffer. The forestomach was cut, removed and discarded. The surface epithelia of the glandular portion of the stomach was gently scraped using a scrapper. This layer was discarded and the gastric mucosa was rinsed with the cold mincing buffer. Then the stomach epithelium was carefully scraped 4-5 times with a scrapper. The cell suspension was then strained using a cell strainer and kept on ice until preparation of slides. The number of isolated living cells was determined by the trypan blue dye exclusion test.

Slide preparation:
From each liver and stomach cell suspension an aliquot of 5 uL and 10 uL, respectively were mixed with low melting agar and the cell/agarose suspension was applied to glass microscope slides previously coated with normal melting agarose.

Lyses:
After the agarose gel has solidified, the slides were placed overnight at 2-8 °C, in a lysis solution consisting of high salts and detergents. The Iysing solution was chilled prior to use, primarily to maintain the stability of the agarose gel.

Unwinding:
After cell lysis two slides for each organ/animal were washed with neutralisation buffer and place in the electrophoresis chamber. The chamber was filled with alkaline buffer and the slides remained in the buffer for 20 minutes to allow DNA to unwind.

Electrophoresis:
After alkali unwinding, the single-stranded DNA in the gels was electrophoresed under alkaline conditions to produce comets. The electrophoretic conditions were 0.7 V/cm for 30 minutes at room temperature (15-30°).

Neutralization and dehydration of slides:
After electrophoresis, the alkali in the gels were neutralized by rinsing the slides with neutralisation buffer and then dehydrated with 100% ethanol, air dried and stored at room temperature.

Staining:
Slides were stained with Sybr-gold prior to scoring.

Scoring:
Fifty randomly selected, non-overlapping cells/slide were scored for DNA damage using the Comet Assay IV v.4.11 Perceptive Instruments software.

DNA damage was assessed by the software system by measuring:
- Comet migration: distance from the perimeter of the comet head to the last visible point in the tail
- %Tail DNA: % tail intensity, the % of DNA present in the tail
- Olive tail moment: product of tail length and %DNA in tail
Each slide was also assessed for possible indications of cytotoxicity, i.e. presence of clouds (termed hedgehogs). The rough estimate of the %clouds was recorded for each slide.

DNA damage evaluation:
Median of 100 counts of %tail DNA, Olive tail moment and tail migration were determined and presented for each animal in each treatment group for each organ. The mean and standard deviation of the median values for %tail DNA and Olive tail moment were presented for each treatment group for each organ.

Evaluation criteria:

The test article was considered to induce DNA damage if:
- A least one of the test doses exhibited a statistically significant increase in tail intensity, compared with the concurrent vehicle control
- The increase was dose related
The test article was considered negative in this assay if neither of the above criteria were met and target tissue was confirmed.

Statistics:

Levene's test was performed to determine heterogenous group variances (p<0.05). Where evidence of heterogeneity was present, the data were rank transformed prior to analysis. Linear regression was used to determine the dose response relationship for the significance level (p<0.01).

Results and discussion

Test results

Test results 1

Sex:

male

Genotoxicity:

negative

Remarks:

Liver

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

not determinable because of methodological limitations

Remarks:

No evidence of target organ exposure demonstrated

Test results 2

Sex:

male

Genotoxicity:

negative

Remarks:

Stomach

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Analytical determinations:
Analysis of Y-14877 in the dosing formulations met the acceptance criteria of 85-115% of target concentration and an RSD of <5%. No test article was detected in the vehicle control.

 Comet assay:

1. Clinical observations: No test article related clinical signs of toxicity were observed.

2. Body weight: No test article related effects on body weight were observed.

3. Toxicity: No test article related toxicity, as evident by hedgehog occurrence on comet slides were observed.

4. Clinical chemistry: not undertaken

5. Necropsy and pathology: Both macroscopic and microscopic observations were deemed to be unremarkable.

6. Target tissue exposure: Concurrent target organ (liver) exposure was not demonstrated. Whilst target exposure to the stomach was not demonstrated, it was not considered necessary as the stomach was the first site of contact following oral gavage dosing directly into the stomach.

7. Comet analysis:

- Liver: The presence of hedgehogs was <3% and <4% for the liver at 4 and 24 hour time points, respectively. Group mean %tail intensity and tail moment values for all groups of animals treated with Y-14877 were similar to the group mean vehicle control data. There were no statistically significant differences in %tail intensity (or tail moment values) between treated and the control group at either time point. All individual animal data at all dose levels were consistent with the vehicle control animals and were generally comparable with the laboratory’s historical control observed range (refer to Table CA 7.6.2/03-1).



-Stomach: The presence of hedgehogs was <27% for the stomach at both time points. The increase in hedgehog values is typical for the stomach due to the mechanical harvesting of stomach epithelium cells required (i.e. scraping of the stomach mucosal epithelia, prior to epithelium cell harvesting).. Group mean %tail intensity values for all groups of animals treated with Y-14877 were similar to the group mean vehicle control data. There were no statistically significant differences in %tail between treated and the control group at either time point. All individual animal data at all dose levels were consistent with the vehicle control animals and were generally comparable with the laboratory’s historical control observed range.

 

Group mean tail moment values of animals treated with Y-14877 showed a statistically significant decrease at the 4 h sample time point. Typically, decreases in comet parameters are deemed indicative of a cross-linking effects, however the standard comet assay needs to be modified to detect such effects, with such effects not being robustly determined in a standard assay. The statistically significant decrease in tail moment values were limited to the 4 h sample time point, were not replicated at the 24 h nor replicated in the %tail intensity values and were not dose related. Therefore, it can be concluded that the decrease in tail moment values were deemed not biologically relevant (refer to Table CA 7.6.2/03-2).

 

Cell viability (assessed by trypan blue exclusion) for the test article-treated liver and stomach cells was <97% for both time points.

 The positive controls induced an acceptable increase in %tail intensity in both liver and stomach, thereby demonstrating the sensitivity and specificity of the test system.

Any other information on results incl. tables

Table CA 7.6.2/03-1:
Group mean and individual animal liver data

Dose level (mg/kg bw)	No. of animals	No .of cells scored	Mean tail intensity^a(% ±SD)		Mean tail moment^a(% ±SD)		Mean % hedgehogs
4 hour sample time
0	5	500	1.89 ±1.18		0.42 ±0.23		---^b
500	5	500	1.71 ±0.89		0.39 ±0.20		---^b
1000	5	500	3.42 ±1.84		0.62 ±0.32		---^b
2000	5	500	1.90 ±1.18		0.40 ±0.22		---^b
MMS, 50	5	500	60.74 ±4.46*		23.13 ±4.30*		---^b
24 hour sample time
0	5	500	0.70 ±0.41		0.15 ±0.08		---^b
500	5	500	1.07 ±0.82		0.23 ±0.13		---^b
1000	5	500	0.7 ±0.25		0.17 ±0.04		---^b
2000	5	500	1.38 ±0.42		0.30 ±0.10		---^b
Historical control data ranges for rat liver comet assay data collected between 2004 - 2007
Vehicle	Mean tail intensity(% ±SD)				Olive tail moment (±SD)
	Mean:			4.40 ±4.23	Mean:	1.03 ±1.07
	Observed range:			0.14 – 10.49	Observed range:	0.02 – 3.33
Positive	Mean tail intensity(% ±SD)				Olive tail moment (±SD)
	Mean:			50.13 ±22.18	Mean:	18.94 ±13.01
	Observed range:			16.86 – 78.68	Observed range:	3.08 – 38.82
*: p<0.05, statistically significant increase +ve control: EMS – ethyl methylsulphonate a   median values of each slide calculated. The mean of the slide medians were calculated to give the individual mean animal value. The individual mean animal values were averaged to provide group means b   no numerical values presented for each animal / group

Table CA 7.6.2/03-2:
Group mean and individual animal stomach data

Dose level (mg/kg bw)	No. of animals	No .of cells scored	Mean tail intensity^a(% ±SD)		Mean tail moment^a(±SD)		Mean % hedgehogs
4 hour sample time
0	5	500	24.37 ±3.90		7.54 ±1.41		---^b
500	5	500	17.55 ±3.62		4.89 ±1.14**		---^b
1000	5	500	15.44 ±1.63		3.94 ±0.61**		---^b
2000	5	500	17.47 ±6.02		4.42 ±1.69**		---^b
MMS, 50	5	500	77.59 ±4.47*		40.80 ±5.26*		---^b
24 hour sample time
0	5	500	5.83 ±2.18		1.39 ±0.51		---^b
500	5	500	12.45 ±12.39		3.57 ±4.06		---^b
1000	5	500	10.00 ±5.03		2.32 ±1.09		---^b
2000	5	500	6.48 ±1.75		1.61 ±0.35		---^b
Historical control data ranges for rat liver comet assay data collected between 2004 - 2007
Vehicle	Mean tail intensity(% ±SD)				Olive tail moment (±SD)
	Mean:			14.11 ±7.65	Mean:	4.63 ±3.48
	Observed range:			7.41 – 26.90	Observed range:	1.75 – 11.40
Positive	Mean tail intensity(% ±SD)				Olive tail moment (±SD)
	Mean:			50.15 ±22.99	Mean:	20.06 ±16.86
	Observed range:			30.61 – 75.53	Observed range:	7.22 – 43.24
*: p<0.05, statistically significant increase, **: p<0.05, statistically significant decrease +ve control: MMS – methyl methanesulphonate a   median values of each slide calculated. The mean of the slide medians were calculated to give the individual mean animal value. The individual mean animal values were averaged to provide group means b   no numerical values presented for each animal / group

Applicant's summary and conclusion

Conclusions:

Based on the results of a reliable Comet assay it is concluded that 3-ethylheptamethyltrisiloxane does not induce DNA damage in stomach and liver cells of male rats following a single oral gavage dose of 2000 mg/kg bw/day, the regulatory maximum recommended dose in accordance with currently regulatory guidelines for short-term in vivo toxicity testing. Although systemic exposure was not demonstrated in this study. based on the full data-set it is reasonable to assume that systemic exposure took place.

Executive summary:

In a liver and stomach comet assay, Sprague Dawley rats (5/group) received a single oral dose of Y-14877 suspended in corn oil (employing a dose volume of 10 mL/kg bw). Doses selected for the comet assay were 0, 1000, 1500 and 2000 mg/kg bw. Liver and stomach were sampled at 4 and 24 hours post dose. A further group of animals (5/sex/group) received a single oral dose of methyl methane sulphonate, with liver and stomach sampled at 4 hour post dosing.

 

The presence of hedgehogs was <3% and <4% for the liver at 4 and 24 hour time points, respectively. Group mean %tail intensity and tail moment values for all groups of animals treated with Y-14877 were similar to the group mean vehicle control data. There were no statistically significant differences in %tail intensity (or tail moment values) between treated and the control group at either time point. All individual animal data at all dose levels were consistent with the vehicle control animals and were generally comparable with the laboratory’s historical control observed range

 

The presence of hedgehogs was <27% for the stomach at both time points. The increase in hedgehog values is typical for the stomach due to the mechanical harvesting of stomach epithelium cells required (i.e. scraping of the stomach mucosal epithelia, prior to epithelium cell harvesting).. Group mean %tail intensity values for all groups of animals treated with Y-14877 were similar to the group mean vehicle control data. There were no statistically significant differences in %tail between treated and the control group at either time point. All individual animal data at all dose levels were consistent with the vehicle control animals and were generally comparable with the laboratory’s historical control observed range.

 

Group mean tail moment values of animals treated with Y-14877 showed a statistically significant decrease at the 4 h sample time point. Typically, decreases in comet parameters are deemed indicative of a cross-linking effects, however the standard comet assay needs to be modified to detect such effects, with such effects not being robustly determined in a standard assay. The statistically significant decrease in tail moment values were limited to the 4 h sample time point, were not replicated at the 24 h nor replicated in the %tail intensity values and were not dose related. Therefore, it can be concluded that the decrease in tail moment values were deemed not biologically relevant (refer to Table CA 7.6.2/03-2).

 

Cell viability (assessed by trypan blue exclusion) for the test article-treated liver and stomach cells was <97% for both time points.

 

The positive controls induced an acceptable increase in %tail intensity in both liver and stomach, thereby demonstrating the sensitivity and specificity of the test system.

It is concluded that Y-14877 did not induce DNA damage in the stomach of male rats following a single oral gavage dose, with harvesting of stomach tissue at 4 and 24 hours later. The maximum dose administered was 2000 mg/kg bw/day, the regulatory maximum recommended dose in accordance with currently regulatory guidelines for short-termi n vivo toxicity testing.

 

Although systemic exposure was not demonstrated in this study. based on the full data-set it is reasonable to assume that systemic exposure took place.