9-[2-(2-methoxyethoxy)ethoxy]-9-[3-(oxiranylmethoxy)propyl]-2,5,8,10,13,16-hexaoxa-9-silaheptadecane

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 June 2017

Reliability:

1 (reliable without restriction)

Justification for type of information:

The absorption, distribution, metabolism and excretion of 9-[2-(2-methoxyethoxy)ethoxy]-9-[3-(oxiranylmethoxy)propyl]-2,5,8,10,13,16-hexaoxa-9-Silaheptadecane have been predicted based on the physico-chemical properties and supporting toxicological information presented for this material.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

assessment report

Reference

Endpoint:

water solubility

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 24 June 2016 and 18 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method A.6 (Water Solubility)

Version / remarks:

EC No. 440/2008 30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 105 (Water Solubility)

Version / remarks:

27 July 1995

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of method:

flask method

Key result

Water solubility:

> 95 vol%

Conc. based on:

test mat.

Loading of aqueous phase:

174 000 mg/L

Temp.:

20 °C

pH:

ca. 7

Key result

Water solubility:

19 136 147 mg/L

Temp.:

20 °C

pH:

7

Details on results:

The standard EC method A6 and OECD method 105 was not applicable to this test item due to the high indeterminable saturation levels produced. It was therefore not possible to prepare samples at five times the saturation level as recommended in the guidelines. No analysis could be performed for the main test due to the high solubility producing unfilterable mixtures. Therefore the water solubility was determined based on visual inspection.
Due to the fast hydrolysis of the test item, a short shake flask was used to confirm the result. The 1 hour timing provided the same result as the 72 hour shake at elevated temperatures. Hydrolysis at pH 7 shows that > 90% of the test item would be remaining with a 1 hour shake at 20 °C and time for the observations.

The results of the definitive test for the water solubility are shown in the table below

Table: Results of Definitive Water Solubility Tests

Concnetration (%w/w)	Observation
4.96	Clear colorless solution free from any excess test item
49.8	Clear colorless solution free from any excess test item
74.8	Clear colorless solution free from any excess test item
89.8	Clear colorless solution free from any excess test item
95.0	Clear colorless solution free from any excess test item
95.0 (1 hour shake)	Clear colorless solution, soingle phase, free from any excess test item

Water solubility greater than 95% w/w at 20.0 ± 0.5 ºC

Conclusions:

The water solubility of the test item has been determined to be greater than 95% w/w of solution at 20.0 ± 0.5 ºC.

Executive summary:

The general physico-chemical properties of 9-[2-(2-METHOXYETHOXY)ETHOXY]-9-[3(OXIRANYLMETHOXY)PROPYL]-2,5,8,10,13,16-HEXAOXA-9-SILAHEPTADECANE have been determined.  

Water Solubility.

Greater than 95% w/w of solution at 20.0 ± 0.5 °C, using the adapted flask method, designed to be compatible with Method A.6 Water Solubility of Commission Regulation (EC) No 440/2008 of 30 May 2008 and Method 105 of the OECD Guidelines for Testing of Chemicals, 27 July 1995.

Cross-reference 2

Reason / purpose for cross-reference:

assessment report

Reference

Endpoint:

partition coefficient

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 24 June 2016 and 18 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method A.8 (Partition Coefficient - HPLC Method)

Version / remarks:

EC No. 440/2008 30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 117 (Partition Coefficient (n-octanol / water), HPLC Method)

Version / remarks:

13 April 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of method:

HPLC method

Partition coefficient type:

octanol-water

Analytical method:

high-performance liquid chromatography

Key result

Type:

Pow

Partition coefficient:

> 2 - < 3.22

Temp.:

20 °C

pH:

5 - 6

Key result

Type:

log Pow

Partition coefficient:

> 0.3 - < 0.507

Temp.:

20 °C

pH:

5 - 6

Details on results:

The predicted partition coefficient determined that a flask method was the most suitable method of analysis, however due to the fast hydrolysis of the test item this method was deemed not suitable and therefore HPLC was conducted. The HPLC method uses a range of standards and as such a limit value was employed on most of the peaks obtained as their retention time was lower than the lowest calibration standard. Limit value agreed with sponsor due to the fast hydrolysis of the test item.

 Preliminary Estimate

The Log₁₀ Pow was calculated to be:       -2.16

Definitive Test

Calibration

The retention times of the dead time and the retention times, capacity factors (k') and log10 Pow values for the reference standards are shown in the following tables:

Dead time	Retention Time (mins)		Mean retention time (mins)
	Injection 1	Injection 2
Thiourea	1.379	1.377	1.378

             

Standard	Retenton time (mins)			Capacity factor (k')	Log10 k'	Log10 Pow
	Injection 1	Injection 2	Mean
2 -Butanone	1.775	1.775	1.775	0.288	-0.541	0.3
Benzonitrile	2.288	2.288	2.288	0.660	-0.180	1.6
Benzene	3.118	3.116	3.117	1.26	0.101	2.1
Naphthalene	4.91	4.910	4.910	2.56	0.409	3.6
Triphenylamine	18.913	18.905	18.909	12.7	1.10	5.7
DDT	27.724	24.709	27.716	19.1	1.28	6.5

Partition coefficient of sample

The retention times, capacity factor and log10 Pow value determined for the sample are shown in the following table:

Peak ID	Injection	Retention time (mins)	Capacity factor k'	Log 10 k'	Log10 Pow	% Area
1	1	1.345	-2.42 x 10^-2	-	>0.3	29.9
	2	1.345	-2.42 x 10^-2	-	>0.3
2	1	1.424	3.33 x 10 ^-2	-1.48	>0.3	38.8
	2	1.424	3.33 x 10^-2	-1.48	>0.3
3	1	1.580	0.147	-0.833	>0.3	1.64
	2	1.582	0.148	-0.829	>0.3
4	1	1.854	0.346	-0.462	0.507	269.7
	2	1.854	0.346	-0.462	0.507

Range log10 Pow:       <0.3 to 0.507

Partition coefficient:       <2.00 to 3.22

Conclusions:

The partition coefficient of the test item has been determined to be in the range of less than 2.00 to 3.22, log10 Pow less than 0.3 (70.3% area) to 0.507 (29.7% area) at 20.0 ±0.5 °C.

Executive summary:

The general physico-chemical properties of 9-[2-(2-METHOXYETHOXY)ETHOXY]-9-[3(OXIRANYLMETHOXY)PROPYL]-2,5,8,10,13,16-HEXAOXA-9-SILAHEPTADECANE have been determined.  

Partition Coefficient (n-octanol/water).

In the range of less than 2.00 to 3.22, log10 Pow <0.3 (70.3% area) to 0.507 (29.7% area) at 20.0 ± 0.5 °C, using the HPLC method, designed to be compatible with Method A.8 Partition Coefficient of Commission Regulation (EC) No 440/2008 of 30 May 2008 and Method 117 of the OECD Guidelines for Testing of Chemicals, 13 April 2004.

Cross-reference 3

Reason / purpose for cross-reference:

assessment report

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 24 June 2016 and 18 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.7 (Degradation: Abiotic Degradation: Hydrolysis as a Function of pH)

Version / remarks:

EC No. 440/2008 30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

Version / remarks:

13 April 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

no

Analytical monitoring:

yes

Buffers:

Specification of Buffer Solutions

Buffer solution(pH) Components Concentration(mol dm-3)
4 Potassium hydrogen phthalate 0.005
7 Disodium hydrogen orthophosphate (anhydrous) 0.003
Potassium dihydrogen orthophosphate 0.002
Sodium chloride 0.002
9 Disodium tetraborate 0.001
Sodium chloride 0.002
These solutions were subjected to ultrasonication and degassing with nitrogen to minimize dissolved oxygen content, all solutions cooled to test temperature before preparation of test solutions.

Details on test conditions:

Performance of the Test

Preparation of the Test Solutions
Sample solutions were prepared in stoppered glass flasks at a nominal concentration of 5.0 g/L in the three buffer solutions.
The solutions were shielded from light whilst maintained at the test temperature.
Tier 2
Results from the method development showed it was necessary to undertake further testing carried out as outlined in the following table:

pH Temperature Time Period (hours)
4 10.0 ± 0.5 °C 1.02
4 20.0 ± 0.5 °C 0.68
4 30.0 ± 0.5 °C 0.42
7 10.0 ± 0.5 °C 61.0
7 20.0 ± 0.5 °C 31.1
7 30.0 ± 0.5 °C 12.2
9 10.0 ± 0.5 °C 3.22
9 20.0 ± 0.5 °C 1.61
9 30.0 ± 0.5 °C 1.10

Positive controls:

no

Negative controls:

no

Preliminary study:

Preliminary Test/Tier 1
Tier 1 testing was not carried out as fast hydrolysis (<7 hours with 50% solvent present) of the sample was seen during the development of the method. Therefore testing was carried out at Tier 2 only.

Transformation products:

not measured

Details on hydrolysis and appearance of transformation product(s):

Identification of Hydrolysis Products – Tier 3
Usually, hydrolysis products should be identified using LC-MS or GC-MS. However, this procedure implies sufficient separation of the individual components that are present in the incubated test item solution (i.e. parent compound and hydrolysis products).
The chromatography of the incubated test item solution resulted in several peaks that could not be separated sufficiently from each other. Thus, identification of the hydrolysis products was technically not feasible. The chemical structure of the hydrolysis product(s) is most probably quite similar to the one of the parent compound.
Analysis of the hydrolyzed test solutions was further hampered due to the fast hydrolysis of the test item. The test item showed an increase and then decrease of the peaks at 1.2 and 2.0 mins as the main peak at 2.4 mins decreased. This would suggest a two fold hydrolysis as the primary break down product forms a secondary breakdown product.
Analysis of the structure of the main constituent could propose that the primary break down produce would be the cleaving of the epoxide group, the weakest link of the structure and prone cleaving under harsher pH conditions.

% Recovery:

ca. 12.1

pH:

4

Temp.:

10.4 °C

Duration:

<= 1.02 h

% Recovery:

ca. 8.31

pH:

4

Temp.:

20.4 °C

Duration:

ca. 0.68 h

% Recovery:

ca. 5.72

pH:

4

Temp.:

30.2 °C

Duration:

ca. 0.42 h

% Recovery:

ca. 47.7

pH:

7

Temp.:

10.4 °C

Duration:

ca. 61 h

% Recovery:

ca. 28.4

pH:

7

Temp.:

20.4 °C

Duration:

ca. 31.3 h

% Recovery:

ca. 26.6

pH:

7

Temp.:

30.2 °C

Duration:

<= 12.24 h

% Recovery:

ca. 18.4

pH:

9

Temp.:

10.2 °C

Duration:

ca. 3.22 h

% Recovery:

ca. 14

pH:

9

Temp.:

20.4 °C

Duration:

ca. 1.61 h

% Recovery:

ca. 7.02

pH:

9

Temp.:

30.4 °C

Duration:

ca. 1.1 h

Key result

pH:

4

Temp.:

25 °C

Hydrolysis rate constant:

ca. 4.9 h-1

DT50:

ca. 0.142 h

Type:

(pseudo-)first order (= half-life)

Key result

pH:

7

Temp.:

25 °C

Hydrolysis rate constant:

ca. 0.058 h-1

DT50:

ca. 12 h

Type:

(pseudo-)first order (= half-life)

Key result

pH:

9

Temp.:

25 °C

Hydrolysis rate constant:

ca. 1.8 h-1

DT50:

ca. 0.385 h

Type:

(pseudo-)first order (= half-life)

Details on results:

Validation
The linearity of the detector response with respect to concentration was assessed over the nominal concentration range of 500 to 7500 mg/L, with standards prepared in acetonitrile. The results were satisfactory with correlation coefficients (r) of ≥ 0.9998 being obtained.

Discussion
During method development and validation the test item was observed to hydrolyze quickly and could be analyzed directly in the buffer solutions without further sample treatment, the rates were calculated by plotting the logarithm of the measured peak areas rather than the concentrations of the test solutions. This is regarded as acceptable as the linearity assessment showed that the measured areas are directly proportional to the concentration of test item. In addition the repeatability assessment showed there was negligible error in the measured areas arising from the analytical methodology. Therefore a plot of log10 area against time has the same slope as a plot of log10 concentration, and hence gives the same calculated rate constant.
The incubation device used during the testing had a limited operating range (nominal 5 °C to 40 °C) and the set points were in 1 °C increments. However the actual test temperatures were recorded to a suitable accuracy with a calibrated thermocouple and thus can be used to suitably model the relationship between temperature and reaction rate.
The nominal 30 °C test at pH 4 hydrolyzed too rapidly to obtain the 6 data points within 90 to 10% hydrolysis. However as the plots of the available points for this test and the subsequent Arrhenius plots both showed good correlation this was considered not to have impacted the result.
The kinetics of the study have been determined to be consistent with that of a pseudo-first order reaction as the graphs of log10 area versus time are straight lines.
No significant peaks were observed at the approximate retention time of the test item on analysis of any matrix blank solutions.
It has been observed that the rate of hydrolysis increases with a move away from neutrality.

The estimated rate constant and half-life at 25 °C of the test item are shown in the following table:

pH	Estimated rate constant (hr-1) at 25 °C	Estimated half-life at 25 °C (hr)
4	4.90	0.142
7	0.0576	12.0
9	1.80	0.385

Validity criteria fulfilled:

yes

Conclusions:

The estimated rate constant and half-life at 25 °C of the test item are shown in the following table:

pH Estimated rate constant (hr-1) at 25 °C Estimated half-life at 25 °C (hr)
4 4.90 0.142
7 5.76 x 10-2 12.0
9 1.80 0.385

Executive summary:

The general physico-chemical properties of 9-[2-(2-METHOXYETHOXY)ETHOXY]-9-[3(OXIRANYLMETHOXY)PROPYL]-2,5,8,10,13,16-HEXAOXA-9-SILAHEPTADECANE have been determined.

Abiotic Degradation, Hydrolysis as a Function of pH.

Assessment of hydrolytic stability was carried out using a procedure designed to be compatible with Method C.7 Abiotic Degradation, Hydrolysis as a Function of pH of Commission Regulation (EC) No 440/2008 of 30 May 2008 and Method 111 of the OECD Guidelines for Testing of Chemicals, 13 April 2004. The results are as follows:

pH	Estimated rate constant (hr-1) at 25 °C	Estimated half-life at 25 °C (hr)
4	4.90	0.142
7	0.0576	12.0
9	1.80	0.385

Cross-reference 4

Reason / purpose for cross-reference:

assessment report

Reference

Endpoint:

adsorption / desorption: screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 06 October 2016 and 27 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.19 (Estimation of the Adsorption Coefficient (KOC) on Soil and Sewage Sludge Using High Performance Liquid Chromatography (HPLC))

Version / remarks:

EC No. 440/2008 30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 121 (Estimation of the Adsorption Coefficient (Koc) on Soil and on Sewage Sludge using High Performance Liquid Chromatography (HPLC))

Version / remarks:

22 January 2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of method:

HPLC estimation method

Radiolabelling:

no

Test temperature:

30”C

Details on study design: HPLC method:

Performance of the Test

Preparation of sample solution
Test item (1.0000 g) was diluted to 20 mL with acetonitrile to give a concentration of 5.00 x 104 g/L.

Preparation of dead time solution
The dead time was determined by measuring the retention time of formamide (purity* 99.94%, 660 mg/L solution in acetonitrile: purified water [55:45 v/v]).

Preparation of reference standard solutions
Solutions of reference standards (see following table) were prepared in methanol.

Standard Purity (%)* Concentration (mg/L)
Acetanilide 99+ 109
Phenol 99.9 119
Atrazine 99.1 96
Isoproturon >98.0 98
Triadimenol 98.0 104
Linuron 99.7 102
Naphthalene 99 99
Endosulfan-diol 99.9 101
Fenthion 97.9 108
-Endosulfan 99.6 105
Phenanthrene 99.1 107
Diclofop-methyl ≥99.5 98
DDT 98.7 100

Determination of retention time
The sample, dead time and reference standard solutions were injected in duplicate using the following HPLC parameters:
HPLC System : Agilent Technologies 1200 Series, incorporating autosampler and workstation
Detector type : Ultraviolet (UV)
Column : Select HSS Cyano 5 m (150 x 4.6 mm id)
Column temperature : 30ºC
Mobile phase : acetonitrile: purified water (55:45 v/v)
pH of mobile phase : 7.4
Flow-rate : 1.0 mL/min
Injection volume : 10 µL
UV detector wavelength : dead time and reference standards: 210 nm
sample: 210 nm

Type:

Koc

Value:

ca. 5.02 - ca. 527 000 dimensionless

pH:

7.4

Temp.:

30 °C

Type:

log Koc

Value:

> 0.701 - < 5.63 dimensionless

pH:

7.4

Temp.:

30 °C

Key result

Type:

log Koc

Value:

1.3 dimensionless

pH:

7.4

Temp.:

30 °C

Remarks on result:

other:

Remarks:

Geometric mean value of four components measured using HPLC

Key result

Type:

Koc

Value:

27.9 dimensionless

Remarks on result:

other:

Remarks:

Geometric mean value of four components measured using HPLC

Details on results (HPLC method):

Calibration
The retention times of the dead time and the retention times, capacity factors (k') and log10 Koc values for the reference standards are shown in the two following tables:
Dead time Retention time (mins) Mean retention time (mins)
Injection 1 Injection 2
Formamide 1.832 1.832 1.832

Standard Retention time (mins) Mean retention time (mins) Capacity factor (k') Log10 k' Log10 Koc
Injection 1 Injection 2
Acetanilide 2.483 2.473 2.478 0.353 -0.452 1.25
Phenol 2.551 2.546 2.549 0.391 -0.408 1.32
Atrazine 3.013 2.998 3.005 0.641 -0.193 1.81
Isoproturon 3.095 3.081 3.088 0.686 -0.164 1.86
Triadimenol 3.324 3.304 3.314 0.809 -9.20 x 10-2 2.40
Linuron 3.586 3.562 3.574 0.951 -2.19 x 10-2 2.59
Naphthalene 3.805 3.776 3.790 1.07 2.90 x 10-2 2.75
Endosulfan-diol 3.533 3.503 3.518 0.920 -3.60 x 10-2 3.02
Fenthion 4.548 4.495 4.521 1.47 0.167 3.31
-Endosulfan 5.408 5.345 5.377 1.94 0.287 4.09
Diclofop-methyl 5.117 5.034 5.075 1.77 0.248 4.20
Phenanthrene 4.354 4.349 4.351 1.38 0.138 4.09
DDT 6.948 6.934 6.941 2.79 0.445 5.63

4.4.4.2       Adsorption Coefficient

The retention times, capacity factor and log10 Koc value determined for the sample are shown in the following table:

Peak	Injection	Retention time (mins)	Capacity factor (k')	Log10 k'	Log10 Koc	Mean log10 Koc	Adsorption Coefficient	Mean % Area
1	1	2.493	0.361	-0.443	0.717	0.701	5.02	37.8
	2	2.483	0.355	-0.449	0.685
2	1	2.784	0.520	-0.284	1.52	1.51	32.6	45.1
	2	2.775	0.515	-0.289	1.50
3	1	3.066	0.674	-0.172	2.10	2.07	116	9.82
	2	3.032	0.655	-0.184	2.04
4	1	8.207	3.48	0.542	5.72	>5.63	>5.27 x 10^5	7.31
	2	8.100	3.42	0.534	5.68

Range log10 Koc:              0.701 to >5.63

Range of Adsorption coefficient:       5.02 to > 5.27 x 10⁵

Area normalisation: 92.7% area has log₁₀Koc 0.701 to 2.07, adsorption coefficient 5.02 to 116

Validity criteria fulfilled:

yes

Conclusions:

The adsorption coefficient (Koc) of the test item has been determined to be a range from 5.02 to greater than 5.27 x 105, log10 Koc from 0.701 to greater than 5.63. 92.7% area has log10Koc 0.701 to 2.07, adsorption coefficient 5.02 to 116.

Executive summary:

The general physico-chemical properties of 9-[2-(2-METHOXYETHOXY)ETHOXY]-9-[3(OXIRANYLMETHOXY)PROPYL]-2,5,8,10,13,16-HEXAOXA-9-SILAHEPTADECANE have been determined.  

Adsorption Coefficient.

5.02 to greater than 5.27 x 10⁵, log₁₀ Koc 0.701 to greater than 5.63, 92.7% area has log₁₀Koc 0.701 to 2.07, adsorption coefficient 5.02 to 116, using the HPLC screening method, designed to be compatible with Method 121 of the OECD Guidelines for Testing of Chemicals, 22 January 2001 and Method C.19 Adsorption Coefficient of Commission Regulation (EC) No 440/2008 of 30 May 2008.

Cross-reference 5

Reason / purpose for cross-reference:

assessment report

Reference

Endpoint:

flash point of flammable liquids

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted on 19 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method A.9 (Flash-Point)

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 440/2008

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of method:

equilibrium method closed cup

Flash point apparatus:

Setaflash apparatus

Key result

Flash point:

ca. 103.3 °C

Atm. press.:

100.2 kPa

Remarks on result:

no flash point up to 100°C

Interpretation of results:

GHS criteria not met

Conclusions:

The flash point of the test item has been determined to be 103 ± 2 °C.

Executive summary:

The flash point of 9-[2-(2-METHOXYETHOXY)ETHOXY]-9-[3(OXIRANYLMETHOXY)PROPYL]-2,5,8,10,13,16-HEXAOXA-9-SILAHEPTADECANE has been determined to be 103 ± 2 °C, using a closed cup equilibrium method designed to be compatible with Method A.9 Flash Point of Commission Regulation (EC) No 440/2008 of 30 May 2008.

Cross-reference 6

Reason / purpose for cross-reference:

assessment report

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

100, 333, 1000, 3333 and 5000µg per plate

Vehicle / solvent:

Water

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535 and TA1537 as described by Ames et al. (1975) and Escherichia coli WP2 uvrA as described by Green and Muriel (1976).
Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Specificity of the reversion mechanism in E. coli is sensitive to basepair substitution mutations, rather than frameshift mutations (Green and Muriel, 1976).
Salmonella tester strains were derived from Dr. Bruce Ames’ cultures; E. coli tester strains were from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No confirmatory (independent repeat) assay was conducted because the results were clearly positive, hence no further testing was warranted.

Conclusions:

All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, 9-[2-(2-methoxyethoxy)ethoxy]-9-[3-(oxiranylmethoxy)propyl]-2,5,8,10,13,16-hexaoxa-9-silaheptadecane did cause a positive mutagenic response with tester strains TA100 and TA1535 in the presence or absence of S9 activation. The study was concluded to be positive without conducting a confirmatory (independent repeat) assay because the results were clearly positive; hence, no further testing was warranted.

Executive summary:

The test substance, 9-[2-(2-methoxyethoxy)ethoxy]-9-[3-(oxiranylmethoxy)propyl]-2,5,8,10,13,16-hexaoxa-9-silaheptadecane, was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system. Water was used as the vehicle.

 

In the preliminary toxicity assay, the dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 μg per plate. Neither precipitate nor background lawn toxicity was observed. Increases in revertant counts were observed with tester strains TA100 and TA1535 in the presence and absence of S9 activation that suggest the presence of mutagenic activity. Based upon these results, the maximum dose tested in the mutagenicity assay was 5000 μg per plate.

 

In the mutagenicity assay, the dose levels tested were 100, 333, 1000, 3333 and 5000 μg per plate. Neither precipitate nor background lawn toxicity was observed. Positive mutagenic responses were observed (3.1- to 14.8-fold, maximum increase) with tester strains TA100 and TA1535 in the presence and absence of S9 activation.

 

These results indicate 9-[2-(2-methoxyethoxy)ethoxy]-9-[3-(oxiranylmethoxy)propyl]-2,5,8,10,13,16-hexaoxa-9-silaheptadecane was positive for the ability to induce reverse mutations at selected loci of two strains of Salmonella typhimurium TA100 and TA1535 in the presence and absence of an exogenous metabolic activation system.

Cross-reference 7

Reason / purpose for cross-reference:

assessment report

Reference

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 July 2016 to 05 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Study was conducted in accordance with OECD 473 and Good Laboratory Practice

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell transformation assay

Specific details on test material used for the study:

Batch/ Lot Number: ADF0041200
Purity: Ca. 81%
Molecular Weight: 500.65 g/mol

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Chinese hamster ovary (CHO-K1) cells (repository number CCL 61)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 was used as the metabolic activation system.

Test concentrations with justification for top dose:

CHO cells were exposed to vehicle alone and to nine concentrations of test substance with half-log dose spacing using single cultures. Precipitation of test substance dosing solution in the treatment medium was determined
using unaided eye at the beginning and conclusion of treatment. The osmolality in treatment medium of the vehicle and that of the highest dose was measured. Doses for the definitive assay were based upon post-treatment toxicity (reduction in cell growth index relative to the vehicle control).Chromosomal Aberration Assays
Based on the results of the preliminary toxicity test, the doses selected for testing in the chromosomal aberration assay were as follows:


Doses (μg/mL)

Non-activated
4wei hr: 5, 15, 50, 150, 300, 450, 600, 700, 800, 1000, 1200
20 hr: 1, 5, 15, 50, 150, 300, 450, 500, 600, 700

S9-activated
4 hr: 0.05, 1, 2.5, 5, 15, 20, 25, 30, 50

Vehicle / solvent:

Water was used as the vehicle based on the solubility of the test substance, and compatibility with the target cells. In a solubility test conducted at BioReliance, the test substance was soluble in water at a concentration of approximately 50 mg/mL, the maximum concentration tested for solubility.

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

The in vitro mammalian chromosomal aberration assay was conducted by exposing CHO cells to appropriate concentrations of the test substance as well as the concurrent positive and vehicle controls, in the presence and absence of an exogenous metabolic activation system. Chinese hamster ovary (CHO-K1) cells (repository number CCL 61) were obtained from American Type Culture Collection, Manassas, VA. In order to assure the karyotypic stability of the cell line, working cell stocks were not used beyond passage 15. The frozen lot of cells was tested using the Hoechst staining procedure and found to be free of mycoplasma contamination. This cell line has an average cell cycle time of 10-14 hours with a modal chromosome number of 20. The use of CHO cells has been demonstrated to be an effective method of detection of chemical clastogens (Preston et al., 1981).

Evaluation criteria:

The test substance was considered to have induced a positive response if
• at least one of the test concentrations exhibits a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), and
• the increase is concentration-related (p ≤ 0.05), and
• results are outside the 95% control limit of the historical negative control data.
The test substance was considered to have induced a clear negative response if none of the criteria for a positive response were met.

Statistics:

Statistical analysis was performed using the Fisher's exact test (p ≤ 0.05) for a pairwise comparison of the frequency of aberrant cells in each treatment group with that of the vehicle control. The Cochran-Armitage trend test was used to assess dose-responsiveness.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

Under the conditions of the assay described in this report, [μ-(5-amino-1,3,3-trimethylcyclohexylamine-N:N’)] hexafluorodiboron was concluded to be negative for the induction of structural and numerical chromosomal aberrations in the non-activated and S9-activated test systems in the in vitro mammalian chromosomal aberration assay using CHO cells.

Executive summary:

The test substance, [μ-(5-amino-1,3,3-trimethylcyclohexylamine-N:N’)] hexafluorodiboron, was tested to evaluate the potential to induce structural chromosomal aberrations using Chinese hamster ovary (CHO) cells in both the absence and presence of an of an exogenous metabolic activation system. CHO cells were treated for 4 hours in the absence and presence of S9, and for 20 hours in the absence of S9.

Water was used as the vehicle.

In the preliminary toxicity assay, the doses tested ranged from 0.2 to 2000 μg/mL, which was the limit dose for this assay. Cytotoxicity (≥ 50% reduction in cell growth index relative to the vehicle control) was observed at 2000 μg/mL in the non-activated 4-hour exposure group; at doses ≥ 60 in the S9-activated 4-hour exposure group; and at doses ≥ 600 μg/mL in the non-activated 20-hour exposure group. Based upon these results, the doses chosen for the chromosomal aberration assay ranged from 500 to 2000 μg/mL for the non-activated 4-hour exposure group; from 5 to 60 μg/mL for the S9-activated 4-hour exposure group; and from 100 to 550 μg/mL for the non-activated 20-hour exposure group.

In the chromosomal aberration assay, cytotoxicity (55 ± 5% reduction in cell growth index relative to the vehicle control) was observed at doses ≥ 1500 μg/mL in the non-activated 4-hour exposure group; at doses ≥ 20 μg/mL in the S9-activated 4-hour exposure group; and at doses ≥ 350 μg/mL in the non-activated 20-hour exposure group. The doses selected for evaluation of chromosomal aberrations were 500, 1000, and 1500 μg/mL for the non-activated 4-hour exposure group; 10, 15, and 20 μg/mL for the S9-activated 4-hour exposure group; and 100, 200, and 350 μg/mL for the non-activated 20-hour exposure group.

In the non-activated 4-hour exposure group, statistically significant increases (2.3% and 1.7%) in structural aberrations was observed at doses 1000 and 1500 μg/mL, respectively (p ≤ 0.05 or p ≤ 0.01; Fisher’s Exact test). Statistically significant increases (3.0% and 3.7%) in numerical aberrations was also observed at doses 500 and 1000 μg/mL, respectively (p ≤ 0.05 or p ≤ 0.01; Fisher’s Exact test). However, the Cochran-Armitage test was negative for a dose response (p > 0.05) for structural and numerical chromosomal aberrations. In addition, the increases in structural and numerical chromosomal aberrations were within their respective historical 95% control limits. Therefore, the statistically significant increases were considered to be biologically irrelevant.

In the S9-activated 4-hour exposure group, no significant or dose-dependent increases in structural aberrations were observed at any dose (p > 0.05; Fisher’s Exact and Cochran-Armitage tests). A statistically significant increase (4.0%) in numerical aberrations was observed at 10 μg/mL (p ≤ 0.01; Fisher’s Exact test). However, the Cochran-Armitage test was negative for a dose response (p > 0.05). In addition, the increase in numerical chromosome aberrations was within the historical 95% control limit. The statistically significant induction was most likely due to low background aberration in concurrent solvent controls. Therefore, the statistically significant increase was considered to be biologically irrelevant.

In the non-activated 20-hour exposure group, no significant or dose-dependent increases in structural or numerical aberrations were observed at any dose (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

The results for the positive and vehicle controls indicate that all criteria for a valid assay were met.

Under the conditions of the assay described in this report, [μ-(5-amino-1,3,3-trimethylcyclohexylamine-N:N’)] hexafluorodiboron was concluded to be negative for the induction of structural and numerical chromosomal aberrations in the non-activated and S9-activated test systems in the in vitro mammalian chromosomal aberration assay using CHO cells.

Cross-reference 8

Reason / purpose for cross-reference:

assessment report

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 01 November 2016 and 15 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Reliability 1 is assigned because the information is based on an acute oral dermal study, which was conducted according to OECD TG 402 in compliance with GLP, without deviations that influence the quality of the results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Version / remarks:

24 February 1987

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.3 (Acute Toxicity (Dermal))

Version / remarks:

EC No. 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

standard acute method

Limit test:

yes

Specific details on test material used for the study:

Identification: 9-[2-(2-METHOXYETHOXY)ETHOXY]-9-[3(OXIRANYLMETHOXY)PROPYL]-2,5,8,10,13,16-HEXAOXA-9-SILAHEPTADECANE
Batch: ADC393100
Purity: 100% UVCB
Physical state/Appearance: clear colorless liquid
Expiry Date: 17 January 2018
Storage Conditions: room temperature in the dark

Species:

rat

Strain:

Wistar

Remarks:

RccHan:WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal Information
Five male and five female Wistar (RccHan:WIST) strain rats were supplied by Envigo RMS (UK) Limited, Oxon, UK. On receipt the animals were randomly allocated to cages. The females were nulliparous and non pregnant. After an acclimatization period of at least 5 days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card. At the start of the study the animals weighed at least 200 g, and were 8 to 12 weeks of age. The weight variation did not exceed ±20% of the mean weight for each sex.

Animal Care and Husbandry
The animals were housed in suspended solid floor polypropylene cages furnished with woodflakes. The animals were housed individually during the 24 Hour exposure period and in groups of five, by sex, for the remainder of the study. Free access to mains drinking water and food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study. The diet, drinking water and bedding were routinely analyzed and were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
The temperature and relative humidity were set to achieve limits of 19 to 25 C and 30 to 70% respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Details on dermal exposure:

On the day before treatment the back and flanks of each animal were clipped free of hair.
Using available information on the toxicity of the test item, a single group of animals was initially treated as follows:
Dose Level(mg/kg) Specific Gravity Dose Volume (mL/kg) Number of Rats
Male Female
2000 1.075 1.87 5 5

The calculated volume of test item, as received, was applied as evenly as possible to an area of shorn skin (approximately 10% of the total body surface area) using a graduated syringe. A piece of surgical gauze was placed over the treatment area and semi occluded with a piece of self adhesive bandage. The animals were caged individually for the 24 Hour exposure period. Shortly after dosing the dressings were examined to ensure that they were securely in place.
After the 24 Hour contact period the bandage was carefully removed and the treated skin and surrounding hair wiped with cotton wool moistened with distilled water to remove any residual test item. The animals were returned to group housing for the remainder of the study period.
The animals were observed for deaths or overt signs of toxicity 30 minutes, 1, 2 and 4 hours after dosing and subsequently once daily for 14 days.
b

Duration of exposure:

24 Hours

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

5

Control animals:

not required

Details on study design:

On the day before treatment the back and flanks of each animal were clipped free of hair.
The calculated volume of test item, as received, was applied as evenly as possible to an area of shorn skin (approximately 10% of the total body surface area) using a graduated syringe. A piece of surgical gauze was placed over the treatment area and semi occluded with a piece of self adhesive bandage. The animals were caged individually for the 24 Hour exposure period. Shortly after dosing the dressings were examined to ensure that they were securely in place.
After the 24 Hour contact period the bandage was carefully removed and the treated skin and surrounding hair wiped with cotton wool moistened with distilled water to remove any residual test item. The animals were returned to group housing for the remainder of the study period.
The animals were observed for deaths or overt signs of toxicity 30 minutes, 1, 2 and 4 hours after dosing and subsequently once daily for 14 days
After removal of the dressings and subsequently once daily for 14 days, the test sites were examined for evidence of primary irritation and scored according to the scale in Table 1 (below):
Any other skin reactions, if present were also recorded.
Individual body weights were recorded prior to application of the test item on Day 0 and on Days 7 and 14.
At the end of the study the animals were killed by cervical dislocation. All animals were subjected to gross necropsy. This consisted of an external examination and opening of the abdominal and thoracic cavities. The appearance of any macroscopic abnormalities was recorded. No tissues were retained

Data Evaluation
Data evaluations included the relationship, if any, between the exposure of the animal to the test item and the incidence and severity of all abnormalities including behavioral and clinical observations, gross lesions, body weight changes, mortality and any other toxicological effects.
Using the mortality data obtained, an estimate of the acute dermal median lethal dose (LD50) of the test item was made.
The results were also evaluated according to the Globally Harmonized System of Classification and Labelling of Chemicals.

Preliminary study:

No reactions

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

no indication of skin irritation up to the relevant limit dose level

Mortality:

There were no deaths

Clinical signs:

other: No signs of systemic toxicity were noted during the observation period.

Gross pathology:

No abnormalities were noted at necropsy

Other findings:

Dermal Reactions
There were no signs of dermal irritation.

Interpretation of results:

GHS criteria not met

Conclusions:

The acute dermal median lethal dose (LD50) of the test item in the Wistar strain rat was found to be greater than 2000 mg/kg body weight.
The test item does not meet the criteria for classification according to the Globally Harmonized System of Classification and Labelling of Chemicals.

Executive summary:

The study was performed to assess the acute dermal toxicity of the test item in the Wistar strain rat.

Methods

A group of ten animals (five males and five females) was given a single, 24 hour, semi occluded dermal application of the undiluted test item to intact skin at a dose level of 2000 mg/kg body weight.  Clinical signs and body weight development were monitored during the study.  All animals were subjected to gross necropsy.

Results

Mortality.  There were no deaths.

Clinical Observations.  There were no signs of systemic toxicity.

Dermal Irritation.  There were no signs of dermal irritation.

Body Weight.  All animals showed expected gains in body weight.

Necropsy.  No abnormalities were noted at necropsy.

Conclusion

The acute dermal median lethal dose (LD50) of the test item in the Wistar strain rat was found to be greater than 2000 mg/kg body weight.

The test item does not meet the criteria for classification according to the Globally Harmonized System of Classification and Labelling of Chemicals.

Cross-reference 9

Reason / purpose for cross-reference:

assessment report

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 21 September2016 and 25 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)

Version / remarks:

2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.1 bis (Acute Oral Toxicity - Fixed Dose Procedure)

Version / remarks:

EC 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

fixed dose procedure

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

RccHan™:WIST strain

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 149 - 178 g
- Fasting period before study: Overnight prior to dosing
- Housing: in groups of up to four in suspended solid floor polypropylene cages furnished with woodflakes
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: >5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25 ”C
- Humidity (%): 30 - 70%
- Air changes (per hr): >15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 21 September 2016 To: 25 October 2016

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

For the purpose of the study the test item was used as supplied. The specific gravity was determined and used to calculate the appropriate dose volume for the required dose level.

Doses:

Using available information on the toxicity of the test item, 2000 mg/kg was chosen as the starting dose. Based on the Specific Gravity (1.075) this resulted in a dose volume of 1.87 mL/kg.

No. of animals per sex per dose:

Preliminary Study = 1 female
Main Study = 4 females

Control animals:

no

Details on study design:

Using available information on the toxicity of the test item, 2000 mg/kg was chosen as the starting dose.
A single animal was treated as follows:
Dose Level (mg/kg) Specific Gravity Dose Volume (mL/kg) Number of Rats
2000 1.075 1.87 1

In the absence of toxicity at a dose level of 2000 mg/kg, an additional group of animals was treated as follows:
Dose Level (mg/kg) Specific Gravity Dose Volume (mL/kg) Number of Rats
2000 1.075 1.87 4

A total of five animals were therefore treated at a dose level of 2000 mg/kg in the study.
All animals were dosed once only by gavage, using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to the fasted body weight at the time of dosing. Treatment of animals was sequential. Sufficient time was allowed between each dose group to confirm the survival of the previously dosed animals.
Clinical observations were made 30 minutes, 1, 2, and 4 hours after dosing and then daily for 14 days. Morbidity and mortality checks were made twice daily, early and late during normal working days, and once daily at weekends and public holidays.
Individual body weights were recorded on Day 0 (the day of dosing) and on Days 7 and 14.
At the end of the observation period the animals were killed by cervical dislocation. All animals were subjected to gross necropsy. This consisted of an external examination and opening of the abdominal and thoracic cavities. The appearance of any macroscopic abnormalities was recorded. No tissues were retained.

Preliminary study:

No toxicity or adverse effects were noted in the preliminary study

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

There were no deaths.

Clinical signs:

other: No signs of systemic toxicity were noted during the observation period

Gross pathology:

No abnormalities were noted at necropsy.

Interpretation of results:

GHS criteria not met

Conclusions:

The acute oral median lethal dose (LD50) of the test item in the female Wistar strain rat was estimated to be greater than 2000 mg/kg body weight (Globally Harmonized Classification System  Unclassified).

Executive summary:

The study was performed to assess the acute oral toxicity of the test item in the Wistar strain rat.

Methods

Following a sighting test at a dose level of 2000 mg/kg, an additional four fasted female animals were given a single oral dose of test item at a dose level of 2000 mg/kg body weight.  Clinical signs and body weight development were monitored during the study.  All animals were subjected to gross necropsy.

Results

Mortality.  There were no deaths.

Clinical Observations.  There were no signs of systemic toxicity.

Body Weight.  All animals showed expected gains in body weight.

Necropsy.  No abnormalities were noted at necropsy.

Conclusion

The acute oral median lethal dose (LD50) of the test item in the female Wistar strain rat was estimated to be greater than 2000 mg/kg body weight (Globally Harmonized Classification System  Unclassified).

Cross-reference 10

Reason / purpose for cross-reference:

assessment report

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Thei study was conducted between 26 October 2016 and 04 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

02 October 2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Version / remarks:

EC No. 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Remarks:

Hsdlf:NZW

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Leicestershire, UK.
- Age at study initiation: 12 - 52 weeks
- Weight at study initiation: 3.72 - 4.07 kg
- Housing: individually housed in suspended cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: >50 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):17 - 23”C
- Humidity (%): 30 - 70%
- Air changes (per hr): >15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 26 October 2016 To: 04 November 2016

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

0.1 mL test material as supplied

Duration of treatment / exposure:

72 hours

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

2

Details on study design:

Immediately before the start of the test, both eyes of the provisionally selected test rabbits were examined for evidence of ocular irritation or defect with the aid of a light source from a standard ophthalmoscope. Only animals free of ocular damage were used.
Initially, a single rabbit was treated. A subcutaneous injection of buprenorphine 0.01 mg/kg was administered 60 minutes prior to test item application to provide a therapeutic level of systemic analgesia. Five minutes prior to test item application, a pre dose anesthesia of ocular anesthetic (two drops of 0.5% proxymetacaine hydrochloride) was applied to each eye.
A volume of 0.1 mL of the test item was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test item, and then released. The left eye remained untreated and was used for control purposes. Immediately after administration of the test item, an assessment of the initial pain reaction was made according to the six point scale shown in Table 1.
Eight hours after test item application, a subcutaneous injection of post dose analgesia, buprenorphine 0.01 mg/kg and meloxicam 0.5 mg/kg, was administered to provide a continued therapeutic level of systemic analgesia. The treated animal was checked for signs of pain and suffering approximately 12 hours later. No further analgesia was required.
After consideration of the ocular responses produced in the first treated animal, a second animal was similarly treated.
Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical evaluation (Draize, J.H, 1977) given in Table 2.
Any other ocular effects were also noted. Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.
Any clinical signs of toxicity, if present, were also recorded.
Individual body weights were recorded on Day 0 (the day of dosing) and at the end of the observation period.

Data Evaluation
The numerical values corresponding to each animal, tissue and observation time were recorded. The data relating to the conjunctivae were designated by the letters A (redness), B (chemosis) and C (discharge), those relating to the iris designated by the letter D and those relating to the cornea by the letters E (degree of opacity) and F (area of cornea involved). For each tissue the score was calculated as follows:
Score for conjunctivae = (A + B + C) x 2
Score for iris = D x 5
Score for cornea = (E x F) x 5

Using the numerical data obtained a modified version of the system described by Kay J.H. and Calandra J.C. (1962) was used to classify the ocular irritancy potential of the test item. This was achieved by adding together the scores for the cornea, iris and conjunctivae for each time point for each rabbit. The group means of the total scores for each observation were calculated. The highest of these group means (the maximum group mean score) together with the persistence of the reactions enabled classification of the eye irritancy potential of the test item.
If evidence of irreversible ocular damage is noted, the test item will be classified as corrosive to the eye.
The results were also evaluated according to the Globally Harmonized System of Classification and Labelling of Chemicals.

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

72 h

Score:

0

Max. score:

8

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

72 h

Score:

0

Max. score:

2

Reversibility:

not specified

Remarks on result:

no indication of irritation

Other effects:

None

Ocular Reactions

Individual and group mean scores for ocular irritation are given in Table 3.  

No corneal or iridial effects were noted during the study.

Moderate conjunctival irritation was noted in one treated eye with minimal conjunctival irritation noted in the other treated eye 1 hour after treatment.  Minimal conjunctival irritation was noted in both treated eyes at the 24 Hour observation and persisted in one treated eye at the 48 Hour observation.

One treated eye appeared normal at the 48 Hour observation and the other treated eye appeared normal at the 72 Hour observation.

Body Weight

One animal showed body weight loss and the other animal showed expected gain in body weight during the study.

Table 3: Individual Total Scores and Group Mean Scores for Ocular Irritation

Rabbir Number and sex	Individual Total Scores at:
	1 hour	24 hours	48 hours	72 hours
75610 Female	8	6	4	2
75630 Female	6	4	0	0
Group Total	14	10	4	0
Group Mean Score	7.0	5.0	2.0	0..0

Interpretation of results:

GHS criteria not met

Conclusions:

The test item produced a maximum group mean score of 7.0 and was classified as a mild irritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.
The test item does not meet the criteria for classification according to the Globally Harmonized System of Classification and Labelling of Chemicals

Executive summary:

The study was performed to assess the irritancy potential of the test item to the eye of the New Zealand White rabbit.

Results

A single application of the test item to the non-irrigated eye of two rabbits produced minimal to moderate conjunctival irritation.  One treated eye appeared normal at the 48 Hour observation and the other treated eye appeared normal at the 72 Hour observation.

Conclusion

The test item produced a maximum group mean score of 7.0 and was classified as a mild irritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.

The test item does not meet the criteria for classification according to the Globally Harmonized System of Classification and Labelling of Chemicals.

Cross-reference 11

Reason / purpose for cross-reference:

assessment report

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 26 September 2016 and 18 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

220July 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

EC 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd strain

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V., Inc., Horst, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known:
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 15 - 23g
- Housing: suspended solid floor polypropylene cages furnished with softwood woodflakes.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: >5 days
- Indication of any skin lesions: none

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25”C
- Humidity (%): 30 - 70%
- Air changes (per hr): >15
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 26 September 2016 To: 18 October 2016

Vehicle:

dimethylformamide

Concentration:

Preliminary screening test: 25µL undiluted test item.

Main Test: undiluted test item or 50% or 25% w/v in dimethyl formamaide

No. of animals per dose:

Prelilminary test = 1
Main test = 4 per dose

Details on study design:

Preliminary Screening Test
Using available information regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µL of the undiluted test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily according to the scale included as Table 1 (below). Any clinical signs of toxicity, if present, were also recorded. The body weight of the mouse was recorded on Day 1 (prior to dosing) and on Day 6.
The thickness of each ear was measured using a Mitutoyo 547 300S gauge (Mitutoyo Corporation), pre dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitization.

Main Test

Test Item Administration
Groups of four mice were treated with the undiluted test item or the test item at concentrations of 50% or 25% v/v in dimethyl formamide. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of four mice received the vehicle alone in the same manner.

3H-Methyl Thymidine Administration
Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 3H methyl thymidine (3HTdR: 80 µCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 µCi to each mouse.

Observations
Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
Body Weights: The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

Terminal Procedures

Termination:
Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 mL of PBS was added to the pooled lymph nodes.

Preparation of Single Cell Suspension:
A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200 mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labeled with the study number and dose concentration. The lymph node cell suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The pooled lymph node cells were pelleted at 1400 rpm (approximately 190 g) for 10 minutes.
The pellet was re suspended in 10 mL of PBS and re pelleted. To precipitate out the radioactive material, the pellet was re suspended in 3 mL of 5% Trichloroacetic acid (TCA).

Determination of 3HTdR Incorporation: After approximately 18 hours incubation at approximately 4 ”C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for 10 minutes, re suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by  scintillation counting. The "Poly Q™" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left to stand in darkness for approximately 20 minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately 20 minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).

Data Evaluation
The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).
The test item will be regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non sensitizer".
The results were also evaluated according to the Globally Harmonized Classification System.

Positive control substance(s):

not specified

Key result

Parameter:

SI

Value:

> 1.12 - < 2.27

Remarks on result:

other: Non-sensitizer

Cellular proliferation data / Observations:

CLINICAL OBSERVATIONS: There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

BODY WEIGHTS: Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.

Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration (% v/v) in dimethyl formamide	dpm	dpm/Node^a	Stimulation Index^b	Result
Vehicle	6564.67	820.58	na	na
25	7384.78	923.10	1.12	Negative
50	8123.43	1015.43	1.24	Negative
100	14902.20	1862.78	2.27	Negative

dpm =  Disintegrations per minute

^a =       Disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8 (total number of lymph nodes)

^b =       Stimulation Index of 3.0 or greater indicates a positive result

na  =    Not applicable

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered to be a non-sensitizer under the conditions of the test.
The test item does not meet the criteria for classification according to the Globally Harmonized Classification System

Executive summary:

A study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

Methods

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 100%, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay.  Three groups, each of four animals, were treated with 50 µL (25 µL per ear) of the undiluted test item or the test item as a solution in dimethyl formamide at concentrations of 50% or 25% v/v.  A further group of four animals was treated with dimethyl formamide alone.

Results

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration (%v/v) indimethyl formamide	Stimulation Index	Result
25	1.12	Negative
50	1.24	Negative
100	2.27	Negative

Cross-reference 12

Reason / purpose for cross-reference:

assessment report

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 20 September 2016 and 21 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to OECD guideline 422 without deviation and in compliance with GLP guidelines.

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

22 March 1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Information as provided by the Sponsor.
Identification : 9-[2-(2-METHOXYETHOXY)ETHOXY]-9-[3(OXIRANYLMETHOXY)PROPYL]-2,5,8,10,13,16-HEXAOXA-9-SILAHEPTADECANE
Physical State/Appearance : Clear colorless liquid
Chemical Name : Organosilane
Purity : 60-100%
Batch Number : ADC393100
Label : FK RAM 1087 ADF0021200
Date Received : 07 April 2016
Storage Conditions : Room temperature, in the dark
Expiry Date : 17 January 2018
No correction for purity was made.

Species:

rat

Strain:

Wistar

Remarks:

Wistar Han™:RccHan™:WIST strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal Information
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for six days during which time their health status was assessed. A total of ninety six animals (forty eight males and forty eight females) were accepted into the study. At the start of treatment the males weighed 291 to 340g, the females weighed 199 to 226g, and were approximately twelve weeks old.

Animal Care and Husbandry
Initially, all animals were housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.

The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for paired animals and mated females during gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation. The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively. Short term deviations from these targets were considered not to have affected the purpose or integrity of the study.

IN-LIFE DATES: From: 27 September 2016 To: 20 November 2016

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Distilled Water. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results show the formulations to be stable for approximately three hours when stored at ambient conditions. Formulations were therefore prepared daily.

VEHICLE
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): constant dosage-volume of 5 mL/kg bw/day

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: Up to 2 weeks
- Proof of pregnancy: Vaginal plug and sperm in vaginal smear referred to as day 0 of gestation
- After successful mating each pregnant female was caged individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

SEE "Any other information" Section below

For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Distilled Water. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results show the formulations to be stable for approximately three hours when stored at ambient conditions. Formulations were therefore prepared daily.
As the test item formulations were prepared on a daily basis, samples of five test item formulations were taken and analyzed for concentration of 9-[2-(2-METHOXYETHOXY)ETHOXY]-9-[3(OXIRANYLMETHOXY)PROPYL]-2,5,8,10,13,16-HEXAOXA-9-SILAHEPTADECANE at Envigo Research Limited, Shardlow, UK, Analytical Services. The method used for analysis of formulations is below. In the majority of instances, test item concentrations in the dosing formulations were retrospectively found to be outside the acceptance criteria of ±20%. With the exception of animals dosed at 1000 mg/kg bw/day which were treated at a lower concentration than intended on one occasion and were found to be within the acceptance criteria during one other analysis point. It can be concluded that animals from all dose groups were generally dosed at higher levels than intended throughout the majority of this study. A review of the formulation and analytical data did not identify a specific reason for these results. As the NOAEL for this study was considered to be 1000 mg/kg bw/day (high dose level) it can be concluded that these higher dose levels did not affect the outcome or integrity of this study.

Duration of treatment / exposure:

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females).

Frequency of treatment:

Once a day for the duration of the study.

Details on study schedule:

Chronological Sequence of Study
i. Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
ii. Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioral toxicity.
iii. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iv. Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
v. On completion of the pairing phase (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
vi. Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.
vii. At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
viii. Blood samples were taken from five males from each dose group for hematological and blood chemical assessments on Day 43. The male dose groups were killed and examined macroscopically on Day 44 or 45.
ix. Blood samples were taken from five randomly selected females from each dose group for hematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically. Any female which did not produce a pregnancy was also killed and examined macroscopically.

Remarks:

Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day

No. of animals per sex per dose:

12 per sex per dose of test item plus 12 per sex in control group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were chosen in collaboration with the Sponsor and were based on the results of previous toxicity work, including a Fourteen Day Repeated Dose Oral (Gavage) Range-Finding Toxicity Study (Study Number: CD65DS), where dose levels up to 1000 mg/kg bw/day were well tolerated. The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.

Positive control:

None

Parental animals: Observations and examinations:

Serial Observations
General Observations/Measurements

Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing (except for females during parturition where applicable). All observations were recorded.

Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Body weights were also recorded at terminal kill.

Food Consumption
During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum. Due to an oversight, food consumption measurements were started on Day 3 post partum for the following animals; 23, 39, 42, 45, 71, 87, 89, 92, 93 and 95. This meant that food consumption data was collected for one day only. As such it has been excluded from the mean food consumption calculations as it was considered not to accurately reflect the groups as a whole.
Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

Water Consumption
Water intake was observed daily by visual inspection of water bottles for any overt changes.
Intergroup differences did not indicate any need for more formal gravimetric measurements.

Specialist Evaluations
Functional Observations
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

Behavioral Assessment
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait Hyper/Hypothermia
Tremors Skin color
Twitches Respiration
Convulsions Palpebral closure
Bizarre/Abnormal/Stereotypic behavior Urination
Salivation Defecation
Pilo-erection Transfer arousal
Exophthalmia Tail elevation
Lachrymation
This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.

Functional Performance Tests
Motor Activity. Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time on each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail, 1979).

Forelimb/Hindlimb Grip Strength. An automated meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).

Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988).
The following parameters were observed:
Grasp response Touch escape
Vocalization Pupil reflex
Toe pinch Blink reflex
Tail pinch Startle reflex
Finger approach

Reproductive Performance
Mating
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of estrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.

Pregnancy and Parturition
Each pregnant female was observed at least three times a day (early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition. Observations were carried out at approximately 0830 and as late as possible at weekends and public holidays. The following was recorded for each female:
i. Date of pairing
ii. Date of mating
iii. Date and time of observed start of parturition
iv. Date and time of observed completion of parturition

In-Life Sampling and Analysis
Hematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 42 for males and Day 4 post partum for females). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling.
Hematology
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices - mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic) - Methylene blue stained slides were prepared but reticulocytes were not assessed
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea Inorganic phosphorus (P)
Glucose Aspartate aminotransferase (ASAT)
Total protein (Tot.Prot.) Alanine aminotransferase (ALAT)
Albumin Alkaline phosphatase (AP)
Albumin/Globulin (A/G) ratio (by calculation) Creatinine (Creat)
Sodium (Na+) Total cholesterol (Chol)
Potassium (K+) Total bilirubin (Bili)
Chloride (Cl-) Bile acids
Calcium (Ca++)

Litter observations:

Litter Data
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.
For each litter the following was recorded:
i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii. Sex of offspring on Days 1 and 4 post partum (this was not conducted for litters from females 15, 38, 44, 61 and 94 in error)
iv. Clinical condition of offspring from birth to Day 5 post partum
v. Individual offspring weights on Days 1 and 4 post partum (Day 4 post partum weights were not conducted in error for litters from females 15, 38, 44, 61 and 94, litter weights were calculated retrospectively from this data)

Physical Development
All live offspring were assessed for surface righting reflex on Day 1 post partum.

Postmortem examinations (parental animals):

Necropsy
Adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 44 or 45. Adult females were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of suitable barbiturate agent. Any females which failed to achieve pregnancy were killed on or after Day 25 post coitum.
For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964). The corpora lutea were also counted.
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights
The following organs were dissected free from fat and weighed before fixation from five selected males and five selected females from each dose group. Tissues shown in bold were weighed from all remaining animals:
Adrenals Pituitary (post-fixation)
Brain Prostate and Seminal Vesicles
Epididymides Spleen
Heart Testes
Kidneys Thymus
Liver Thyroid (weighed post-fixation with Parathyroid)
Ovaries Uterus (weighed with Cervix)

Histopathology
Samples of the following tissues were removed from five selected males and five selected females from each dose group and preserved in buffered 10% formalin, except where stated. Tissues shown in bold were preserved from all remaining animals:
Adrenals Muscle (skeletal)
Aorta (thoracic) Ovaries
Bone & bone marrow (femur including stifle joint) Pancreas
Bone & bone marrow (sternum) Pituitary
Brain (including cerebrum, cerebellum and pons) Prostate
Caecum Rectum
Coagulating gland Salivary glands (submaxillary)
Colon Sciatic nerve
Duodenum Seminal vesicles
Epididymides Skin
Esophagus Spinal cord (cervical, mid thoracic
Eyes and lumbar)
Gross lesions Spleen
Heart Stomach
Ileum (including peyer’s patches) Testes
Jejunum Thyroid/Parathyroid
Kidneys Trachea
Liver Thymus
Lungs (with bronchi)# Urinary bladder
Lymph nodes (mandibular and mesenteric) Uterus & Cervix
Mammary gland Vagina
Tissues were dispatched to the Test Site (Envigo CRS Limited, Eye,Suffolk, IP23 7PX) for processing). The tissues from five selected control and 1000 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with hematoxylin and eosin for subsequent microscopic examination. The tissues shown in bold from the remaining control and 1000 mg/kg bw/day animals were also processed. In addition, sections of testes from all control and 1000 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.
Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell-or stage-specificity of testicular findings was noted.
Since there were indications of treatment-related changes in the spleens of females, examination was subsequently extended to include similarly prepared sections of spleens from female animals in the low and intermediate groups.

Pathology
Microscopic examination was conducted by the Study Pathologist (W Henderson). A peer review of the findings observed was conducted by V Mowat at Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS, UK.

Data Evaluation
Data were processed to give summary incidence or group mean values and standard deviations where appropriate. All data were summarized in tabular form.

Statistics:

SEE "Any other information on materials and methods" below

Reproductive indices:

Mating Performance and Fertility
The following parameters were calculated from the individual data during the mating period of the parental generation:
Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
Fertility Indices
Mating index (%) : Number animals mated / Animals paired × 100
Pregnancy Index (%) (%) : Number pregnant females / Number of animals mated × 100
Gestation and Parturition Data
The following parameters were calculated from individual data during the gestation and parturition period of the parental generation:
Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
Parturition Index
The following was calculated for each group:
Parturition Index (%) : Number of females delivering live offspring / Number of pregnant females x100

Offspring viability indices:

The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).
Implantation losses
Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:
Pre–implantation loss (%) : No. corpora lutes - No. implantaion sites / No. corpora lutes x 100
Post-implantation loss (%) : No. implantation sites - total no. offspring born / No. implantation sites x 100
Live Birth and Viability Indices
Live birth index (%) : Number live offspring on Day 1 after littering / Total number of offspring born × 100
Viability index (%) : Number live offspring on Day 4 after littering / Number live offspring on Day 1 after littering × 100
Sex ratio (% males)
Sex ratio was calculated for each litter value on Days 1 and 4 post partum, using the following formula:
Percentage of males : Number of male offspring/ Total number of offspring x 100

Clinical signs:

no effects observed

Description (incidence and severity):

There were generally no findings observed that indicated any adverse systemic toxicity of the test item in animals from any treatment group.
Animals of either sex (12 males and 12 females) treated with 1000 mg/kg bw/day showed incidences of increased salivation from Day 2 until Day 43 (males) and Day 45 (females). Two males treated with 300 mg/kg bw/day exhibited intermittent instances of increased salivation from Days 16 to 43. One female treated with 1000 mg/kg bw/day exhibited noisy respiration on Day 44 only. Such instances of increased salivation are frequently observed when animals are dosed via the oral gavage route and are generally considered to be associated with distaste or slight irritancy of the test item formulations rather than indicating any systemic effect of treatment. Noisy respiration can also be observed following the oral administration of a slightly irritant test item formulation and in isolation was considered not to be of toxicological significance. One female treated with 1000 mg/kg bw/day exhibited pilo-erection from Days 36 to 37. Observations of this nature are not uncommon around the time of parturition and in isolation was considered unrelated to treatment. One female animal treated with 100 mg/kg bw/day exhibited an abnormal gait (unable to put pressure on left forelimb) from Days 7 to 21. This was considered to be the result of a physical injury and unrelated to treatment. One control male animal exhibited noisy respiration on Day 39 and a further control male exhibited generalized fur loss from Days 38 to 45. As these animals were not treated with the test item these findings are of no toxicological significance.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the study

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Male animals from all treatment groups generally exhibited reduced body weight gains during the treatment period resulting in overall reductions in absolute body weight gain in a dose related manner. Reductions in body weight gains achieved statistical significance in male animals treated with 1000 mg/kg bw/day during Weeks 1 and 4 (p<0.05) and Week 6 (p<0.01). Statistical significance was achieved during Week 4 (p<0.01) and during Week 6 (p<0.01) for animals treated with 300 mg/kg bw/day.
There was considered to be no adverse effect on body weight development for female animals treated with 100, 300 or 1000 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was considered to be no significant effect of treatment with the test item up to a dose level of 1000 mg/kg bw/day on dietary intake for animals of either sex throughout the treatment period.
Female animals treated with 1000 mg/kg bw/day exhibited slight reductions in food consumption during the first two weeks of maturation but without achieving statistical significance. This slight reduction in food consumption continued for the first two weeks of gestation, achieving statistical significance (p<0.05) during the first week. No such effects were noted during the lactation phase of the study.
Minor fluctuations were noted in food conversion efficiency, however, these differences were deemed to be reflective of intergroup differences in body weight gains and/or dietary intake.

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

Visual inspection of water bottles didn't reveal any intergroup differences in water consumption

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Female animals treated with 1000 mg/kg bw/day showed statistically significant increases (p<0.01) in mean corpuscular hemoglobin and mean corpuscular volume, increases appeared to be dose related. The majority of animals exhibited values that were within the historical control data range. However, these hematological findings may be linked to the minor increases in splenic hematopoiesis noted in these females, although no such in-life changes were noted in females treated with 100 or 300 mg/kg bw/day. As such, the changes noted in these hematological parameters are considered to be of equivocal significance, and could be considered as non-adverse.
There were considered to be no treatment-related effects detected in the hematological parameters examined for male animals from any treatment group.
Male animals from all dose groups showed statistically significant reductions in mean corpuscular hemoglobin concentration in a dose related manner (p<0.05 (low and intermediate) and p<0.01 (high)). Male animals treated with 1000 mg/kg bw/day showed a statistically significant reduction (p<0.05) in total leukocyte count and lymphocytes and a statistically significant increase (p<0.05) in activated partial thromboplastin time. Eosinophils were statistically significantly reduced (p<0.05) in all treated male animals, however, a true dose-related response was not evident. With the exception of two isolated instances in animals treated with 1000 mg/kg bw/day (one showing decreased mean corpuscular hemoglobin concentration and one exhibiting decreased lymphocytes) all values were within the historical control data range and as there were no corresponding histopathological correlates these findings were considered not to be related to systemic toxicity of the test item.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were considered to be no treatment-related effects detected in the blood chemical parameters examined.
Male animals from all treatment groups showed a statistically significant reduction (p<0.01) in glucose levels. In contrast, blood glucose was statistically significantly higher (p<0.05) in female animals treated with 300 mg/kg bw/day. Chloride concentrations were statistically higher (p<0.05) for male animals treated with 300 mg/kg bw/day and 1000 mg/kg bw/day, although a true dose-related response was not evident.
With the exception of male animals that were treated with 300 mg/kg bw/day which exhibited chloride concentrations which were higher than the historical control data range, the majority of blood chemical values lay within the historical control ranges and as there were no corresponding histopathological correlates these findings were considered to be incidental and unrelated to treatment of the test item.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Behavioral Assessments
There were no treatment-related changes in the behavioural parameters at 100, 300 or 1000 mg/kg bw/day.
Functional Performance Tests
There were no toxicologically significant changes in functional performance considered to be related to treatment at 100, 300 or 1000 mg/kg bw/day.
Females from all treatment groups showed a statistically significant reduction (p<0.05) in hindlimb grip strength. The intergroup differences were confined to one out of the three tests and a true dose-related response was not evident. In the absence of any clinical signs of neurotoxicity evident on the study, the intergroup differences were considered not to be of toxicological importance.
Sensory Reactivity Assessments
There were no inter-group differences in sensory reactivity scores that were considered to be related to treatment at 100, 300 or 1000 mg/kg bw/day.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Spleen
There was a minor increase in hematopoiesis in the spleen of treated female animals. This occurred in two females treated with 100 mg/kg bw/day (minimal), three females treated with 300 mg/kg bw/day (1 minimal, 2 mild) and in four females treated with 1000 mg/kg bw/day (2 minimal, 2 mild). This also occurred at a marked severity in a further female treated with 300 mg/kg bw/day, this was considered to be due to a macroscopic abnormality in the stomach (ulceration).
No other changes were noted to account for findings seen in-life or variations in organ weights.
Non-Productive Mating
The following pairings did not produce a litter despite positive signs of mating:
Animals 17F and 5M (Control) showed no histological changes to account for the lack of pregnancy.
Animals 48F and 36M (Group 2) showed no histological changes to account for the lack of pregnancy.
There were no test item-related microscopic findings in the testes, including following the qualitative examination of the stages of spermatogenesis (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or following the evaluation of the uterus or evaluation of follicles and corpora lutea in the ovaries.

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating
No treatment-related effects were detected in mating performance.

Fertility
No treatment-related effects were detected in fertility.
One control female and one female treated with 100 mg/kg bw/day were not pregnant following positive evidence of mating. No histological changes were noted to account for the lack of pregnancy, therefore this was considered to be incidental.

Gestation Length
Gestation lengths were generally between 22 and 23½ days. One female treated with 100 mg/kg bw/day had a gestation length of 24 days and a female treated with 300 mg/kg bw/day exhibited a gestation length of 24½ days but ultimately had a total litter loss. Mean gestation lengths for females treated with 1000 mg/kg bw/day showed a statistically significant increase (p<0.05) in gestation length when compared to control. However, all gestation lengths were within the historical control range, and as such the intergroup variations were considered not to be of toxicological significance.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Systemic toxicity

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOEL

Effect level:

ca. 10 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

not determinable due to absence of adverse toxic effects

Clinical signs:

no effects observed

Description (incidence and severity):

No obvious clinical signs of toxicity were detected for offspring from treated females when compared to controls. The incidental clinical signs detected throughout the control and treated groups, consisting of small size, weak, no milk in the stomach, physical injury, missing, cannibalized or found dead were considered to be low incidence findings observed in offspring in studies of this type and were considered unrelated to test item toxicity.

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Statistical analysis of the litter, offspring weights or surface righting reflex data did not reveal any significant intergroup differences.
For animals treated with 1000 mg/kg bw/day, actual litter weights were slightly lower than controls on Days 1 and 4 post partum, which is an obvious effect of the slightly lower litter size apparent at this dosage as a result of one female with only three offspring born. Individual male and female mean offspring body weights were generally comparable to control.

Gross pathological findings:

no effects observed

Offspring Litter Size, Sex Ratio and Viability
With the exception of one female treated with 1000 mg/kg bw/day which only gave birth to three offspring, of the litters born, litter size at birth and subsequently on Days 1 and 4 post partum were comparable to controls.
In general, the number of corpora lutea and implantations were similar across all treatment groups when compared to control and as such there could be considered to be no significant treatment-related effects. Statistical analysis of the data did not reveal any significant intergroup differences. However, animals treated with 1000 mg/kg bw/day exhibited an increase in both pre and post-implantation losses and a slightly lower litter size (but without achieving statistical significance in any parameter) when compared to control. The post-implanation loss and slightly lower litter sizes were, however, considered to be mainly due to one female animal which only gave birth to three offspring. A further two animals from this dose group also gave birth to only six offspring, however, this litter size is within the historical control data range and as such these differences could be considered to be due to normal biological variation. Offspring viability were comparable to control in all treatment groups. Total litter losses were noted in one control female and another was noted in one female treated with 300 mg/kg bw/day. In the absence of a similar effect seen at 1000 mg/kg bw/day, these total litter losses were considered unrelated to treatment.
There were no intergroup differences in sex ratio (percentage male offspring) for litters from treated groups compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Reproductive effects observed:

no

Conclusions:

The oral administration of 9-[2-(2-METHOXYETHOXY)ETHOXY]-9-[3(OXIRANYLMETHOXY)PROPYL]-2,5,8,10,13,16-HEXAOXA-9-SILAHEPTADECANE to rats by gavage, at dose levels of 100, 300 and 1000 mg/kg bw/day, did not result in any significant adverse toxicological effects. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 1000 mg/kg bw/day.
The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day.

Executive summary:

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development) and is designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 22 March 1996).

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 mg/kg bw/day.  A control group of twelve males and twelve females was dosed with vehicle alone (Distilled water).

Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study.  

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 4 post partum.  Hematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group.  

Adult males were terminated on Day 44 or 45, followed by the termination of all females and offspring on Day 5 post partum.  Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum.  All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results

Adult Responses

Mortality

There were no unscheduled deaths during the study.

Clinical Observations

There were generally no findings observed that indicated any adverse systemic toxicity of the test item in animals from any treatment group.

Behavioral Assessment

There were no treatment-related changes in the behavioural parameters at 100, 300 or 1000 mg/kg bw/day.

Functional Performance Tests

There were no toxicologically significant changes in functional performance considered to be related to treatment at 100, 300 or 1000 mg/kg bw/day.

Sensory Reactivity Assessments

There were no inter-group differences in sensory reactivity scores that were considered to be related to treatment at 100, 300 or 1000 mg/kg bw/day.

Body Weight

Male animals from all treatment groups generally exhibited reduced body weight gains during the treatment period resulting in overall reductions in absolute body weight gain in a dose related manner.  

There was considered to be no adverse effect on body weight development for female animals treated with 100, 300 or 1000 mg/kg bw/day.

Food Consumption

There was considered to be no significant effect of treatment with the test item up to a dose level of 1000 mg/kg bw/day on dietary intake for animals of either sex throughout the treatment period.  Minor fluctuations were noted in food conversion efficiency, however, these differences were deemed to be reflective of intergroup differences in body weight gains and/or dietary intake.

Water Consumption

Visual inspection of water bottles didn't reveal any intergroup differences in water consumption.

Reproductive Performance

Mating

No treatment-related effects were detected in mating performance.

Fertility

No treatment-related effects were detected in fertility.

Gestation Lengths

Gestation lengths were generally between 22 and 23½ days and the distribution of gestation lengths for treated females was essentially similar to control.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

Of the litters born, litter size at birth and subsequently on Days 1 and 4 post partum, sex ratio and viability were generally comparable to controls.

Offspring Growth and Development

No obvious affect of treatment was noted in offspring growth and development.  In general, offspring body weight gain and litter weights on Days 1 and 4 post partum were comparable to control litters.  Surface righting was also generally comparable to controls.

Laboratory Investigations

Hematology

There were considered to be no treatment-related effects detected in the hematological parameters examined for male animals from any treatment group.

Female animals treated with 1000 mg/kg bw/day exhibited increases in mean corpuscular hemoglobin and mean corpuscular volume.  No such effects were detected in females treated with 300 or 100 mg/kg bw/day.

Blood Chemistry

There were considered to be no treatment-related effects detected in the blood chemical parameters examined.

Pathology

Necropsy

Macroscopic examination at terminal necropsy did not reveal any findings that were considered to be treatment related in animals of either sex up to a dose level of 1000 mg/kg bw/day.

Organ Weights

There were considered to be no treatment-related effects detected in animals of either sex up to a dose level of 1000 mg/kg bw/day.

Histopathology

There was a minor increase in hematopoiesis in the spleen of treated female animals.  This occurred in two females treated with 100 mg/kg bw/day (minimal), three females treated with 300 mg/kg bw/day (1 minimal, 2 mild) and in four females treated with 1000 mg/kg bw/day (2 minimal, 2 mild).

This also occurred at a marked severity in a further female treated with 300 mg/kg bw/day.  This was considered to be a consequence of a separate abnormality (marked ulceration of the stomach).

Conclusion

The oral administration of 9-[2-(2-METHOXYETHOXY)ETHOXY]-9-[3(OXIRANYLMETHOXY)PROPYL]-2,5,8,10,13,16-HEXAOXA-9-SILAHEPTADECANE to rats by gavage, at dose levels of 100, 300 and 1000 mg/kg bw/day, did not result in any significant adverse toxicological effects.  The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 1000 mg/kg bw/day.

The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Objective of study:

absorption

distribution

excretion

metabolism

toxicokinetics

Principles of method if other than guideline:

The absorption, distribution, metabolism and excretion of 9-[2-(2-methoxyethoxy)ethoxy]-9-[3-(oxiranylmethoxy)propyl]-2,5,8,10,13,16-hexaoxa-9-Silaheptadecane have been predicted based on the physico-chemical properties and supporting toxicological information presented for this material.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

9-[2-(2-methoxyethoxy)ethoxy]-9-[3-(oxiranylmethoxy)propyl]-2,5,8,10,13,16-hexaoxa-9-silaheptadecane

EC Number:

289-390-3

EC Name:

9-[2-(2-methoxyethoxy)ethoxy]-9-[3-(oxiranylmethoxy)propyl]-2,5,8,10,13,16-hexaoxa-9-silaheptadecane

Cas Number:

88127-84-8

Molecular formula:

C21H44O11Si

IUPAC Name:

9-[2-(2-methoxyethoxy)ethoxy]-9-{3-[(oxiran-2-yl)methoxy]propyl}-2,5,8,10,13,16-hexaoxa-9-silaheptadecane

Test material form:

liquid

Details on test material:

Batch number: 84246753
Expiry Date: 14 August 2017

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:

Information from single and repeat dose toxicity studies suggest that the gastro-intestinal tract would be the primary route of absorption following oral administration before entering the circulatory system via the blood. The high vapour pressure and flammability values imply the risk of inhalation exposure to volatile products to be minimal and were it to occur, the supporting toxicity studies indicate the risks of accumulative systemic toxicity to be minimal.

Details on distribution in tissues:

The available information relating to the distribution of the test item implies the most probable route of systemic distribution would take place in the circulatory system via the serum. The absence ofinformation to suggest the test item is a skin sensitizer affirms that the test item is unlikely to bind to carrier proteins in the circulatory system.

Details on excretion:

There is no evidence to indicate the route of excretion but high water-soluble products are not favourable for biliary excretion and therefore urinary excretion may well be a significant route for this material. Any test item that is not absorbed would be excreted in the faeces.

Metabolite characterisation studies

Metabolites identified:

no

Details on metabolites:

Genotoxicity assays provided “ p o s i t i v e ” outcomes, which were neither enhanced nor diminished in the presence of the S9 metabolising system. However, in isolation these findings do not necessarily reflect human metabolism and the rodent oral (gavage) reproductive screening (OECD 422) study did not provide any evidence to indicate test item or metabolite influenced hepatic metabolism.

Applicant's summary and conclusion

Conclusions:

The available information suggests that absorption of the test item would take place in the gastrointestinal tract and once absorbed, the test item or metabolites in all probability would be distributed in the serum. . Absorbed test material is then likely to undergo hepatic transformation and the primary route of excretion of the test item and any of its metabolites is expected to be from the urine with any test item that is not absorbed excreted in the faeces.

Executive summary:

The substance composed, as listed in Section 3 is a clear colourless liquid with good water solubility and high melting and auto-ignition points. The supporting physico-chemical propertiesindicate low volatility suggesting the risk of particle inhalation of the test item to be unlikely. Results from genotoxicity assays proved positive for both mutagenicity and clastogenicity. Results from single dose toxicity studies confirmed the oral and dermal LD50 were each greater than 2000 mg/kg body weight and a rodent oral (gavage) reproductive screening (OECD 422) study further confirmed the risk of accumulative systemic toxicity to be low. Local dermal tolerance verified the test item is not

irritant to rabbit skin but is mildly irritant to the rabbit eye. Results from a local lymph node assay indicated the test item did not require classification as a skin sensitizer.

Absorption

Information from single and repeat dose toxicity studies suggest that the gastro-intestinal tract would be the primary route of absorption following oral administration before entering the circulatory system via the blood. The high vapour pressure and flammability values imply the risk of inhalation exposure to volatile products to be minimal and were it to occur, the supporting toxicity studies indicate the risks of accumulative systemic toxicity to be minimal.

Distribution

The available information relating to the distribution of the test item implies the most probable route ofsystemic distribution would take place in the circulatory system via the serum. The absence of information to suggest the test item is a skin sensitizer affirms that the test item is unlikely to bind to carrier proteins in the circulatory system.

Metabolism

Genotoxicity assays provided “ p o s i t i v e ” outcomes, which were neither enhanced nor diminished in the presence of the S9 metabolising system. However, in isolation these findings do not necessarily reflect human metabolism and the rodent oral (gavage) reproductive screening (OECD 422) study did not provide any evidence to indicate test item or metabolite influenced hepatic metabolism.

Excretion

There is no evidence to indicate the route of excretion but high water-soluble products are not favourable for biliary excretion and therefore urinary excretion may well be a significant route for this material. Any test item that is not absorbed would be excreted in the faeces.

CONCLUSION

The available information suggests that absorption of the test item would take place in the gastrointestinal tract and once absorbed, the test item or metabolites in all probability would be distributed in the serum. . Absorbed test material is then likely to undergo hepatic transformation and the primary route of excretion of the test item and any of its metabolites is expected to be from the urine with any test item that is not absorbed excreted in the faeces.