Hexadecyltrimethoxysilane

Long-term toxicity to fish

Administrative data

Endpoint:

fish early-life stage toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

biodegradation in water: ready biodegradability

Remarks:

prolonged to 60 d

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Jul - 24 Sep 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Staatliches Gewerbeaufsichtsamt Hildesheim

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Municipal sewage treatment plant, Hildesheim, Germany
- Pretreatment: Sludge was washed twice with chlorine free tap water. After the second washing settled sludge was resuspended in mineral salts medium and was maintained in an aerobic condition by aeration with CO2 free air until test start (2 d). The amount of inoculum used to initiate inoculation was 3.83 mg/L (25 mg/L dw)
- Initial cell/biomass concentration: Colony Forming Units (CFU) of the inoculum were determined prior to test start by standard dilution plate count. The CFU concentration of the inoculum corresponds to approx. 0.52 x 10^7 CFU/L in the final test solution.
- Water filtered: no

Duration of test (contact time):

60 d

Initial conc.:

20 mg/L

Based on:

test mat.

Initial conc.:

51.2 other: mg O2/L

Based on:

ThOD

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: according to guideline
- Solubilising agent: silicone oil (500 µL in 250 mL test solution, 100 mg test item / 10 mL silicone oil)
- Test temperature: 21 - 21.7 °C
- pH: inoculum control: 7.51 - 7.65, functional control: 7.64 - 7.68, inoculum control with silicone oil: 7.51 - 7.73, test item: 7.5 - 7.71, toxicity control: 7.64 - 7.73
- pH adjusted: no
- Continuous darkness: yes
- Other: continuous stirring

TEST SYSTEM
- Culturing apparatus: 500 mL brown glass bottles filled with 250 mL
- Number of culture flasks/concentration: 2
- Measuring equipment: OxiTop measuring heads
- Test performed in open system: no
- Details of trap for CO2 and volatile organics if used: A rubber sleeve with soda lime was hung into the opening of the bottles to absorb evolved CO2.

SAMPLING
- Sampling frequency and method: The oxygen consumption was determined in the incubation vessels by the OxiTop measuring system at 360 measuring points (every 240 min) during the 60 d incubation period.

CONTROL AND BLANK SYSTEM
- Inoculum control: 2 replicates (test medium without test and / or reference item)
- Inoculum control with silicone oil: 2 replicates (test medium without test and / or reference item and 500 µL silicone oil)
- Toxicity control: 1 replica (test item (incl. 500 µL silicone oil) and reference item)
- Functional control: 1 replica
- Functional control with silicone oil: 1 replica

Reference substance:

benzoic acid, sodium salt

Remarks:

30 mg/L

Reference substance:

benzoic acid, sodium salt

Remarks:

30 mg/L sodium benzoate and 500 µL silicone oil / 250 mL test solution

Parameter:

% degradation (O2 consumption)

Value:

65

Sampling time:

28 d

Parameter:

% degradation (O2 consumption)

Value:

81

Sampling time:

60 d

Details on results:

- The biodegradation of the substance failed the 10 - d window criterion.
- O2 depletion in inoculum control with silicon oil: 28.6 mg O2/L on Day 28 and 33.5 mg/L on Day 60.
- Toxicity control: 47% degradation of test item after 14 d and 86% after 60 d.

Results with reference substance:

- Functional control: 86% biodegradation of sodium benzoate on Day 14.
- Functional control with silicone oil: 98% biodegradation on Day 60.

Table 1: % Biodegradation of test item, functional control and toxicity control during 60 d.

[d]	% Biodegradation
	Functional Control	Functional control with silicon oil	Test Item 1	Test Item 2	Tox. Control
2	30	30	3	1	14
4	64	66	3	3	33
6	75	78	3	3	39
8	83	86	4	4	43
10	84	90	9	9	45
12	85	90	23	20	46
14	86	90	36	34	47
16	81	90	40	41	48
18	78	93	46	45	55
20	77	94	51	52	62
22	78	94	56	57	67
24	78	93	59	60	70
26	78	94	62	64	72
28	79	94	64	66	74
30	80	95	65	68	76
32	80	94	67	70	78
34	80	96	67	72	79
36	79	96	70	74	80
38	77	97	70	74	80
40	80	95	71	76	80
42	81	94	72	78	81
44	80	96	73	79	82
46	82	97	75	79	83
48	82	96	75	80	83
50	81	96	75	80	83
52	80	97	76	80	84
54	80	97	76	81	84
56	78	97	76	81	84
58	79	97	78	81	85
60	77	98	79	83	86

Table 2: Validity criteria for OECD 301 F.

Criterion from the guideline	Outcome	Validity criterion fulfilled
Difference of extremes of replicate values of the removal of the test chemical at the plateau, at the end of the test or at the end of the 10-d window, as appropriate, is less than 20%.	4%	yes
Percentage degradation of the reference compound reached the pass level by day 14 (≥ 60%).	Functional control: 86% Functional control with silicone oil: 90%	yes
The toxicity control should degrade to at least 35% (based on DOC) or at least 25% (based on ThOD or ThCO2) within 14 d.	47%	yes
The oxygen uptake of the inoculum blank is normally 20-30 mg O2/L and should not be greater than 60 mg/L in 28 days.	Functional control: 32% Functional control with silicone oil: 28.6%	yes
the pH value should be in the range 6-8.5.	7.47 – 7.64	yes

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Interpretation of results:

readily biodegradable, but failing 10-day window

Cross-reference 2

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

water solubility

Type of information:

(Q)SAR

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

QSAR prediction

Principles of method if other than guideline:

The result was obtained using an appropriate QSAR method (see QMRF and QPRF for details).

Water solubility:

0.002 mg/L

Temp.:

20 °C

pH:

7

Estimated value, no data available for pH.

Conclusions:

Interpretation of results: insoluble (< 0.1 mg/L)
A solubility of 2.0E-3 mg/l at 20°C was predicted for the substance based on an appropriate calculation method. The result is considered to be reliable.

Cross-reference 3

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1991-06-03 to 1991-06-07

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Study followed a protocol based on the OECD 201 (1984) guideline. It should be noted, that only two parallels were investigated for both control and test series. Thus no thorough statistical assessment of effect values is possible. The study duration was 96 hours which is not in accordance with OECD standards. Thus a recalculation of effect values for 72 h has been performed in order to meet CLP and OECD standards.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

1984

Deviations:

yes

Remarks:

Methanol and tertiary butanol (TBA) were used as solvents to aid test substance dispersion, test duration was 96 hours, only two parallels for both control and test series, respectively.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: Two stock solutions (nominally 300 g/L) were prepared in methanol and tertiary butanol (TBA). The stock solutions were added to the algal culture medium to produce the desired test concentrations.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): It is likely that all exposure concentrations exceeded the solubility of the test substance in the test medium.
- Controls: Algal culture medium

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM

- Strain: SAG 86.81

- Source: Culture supplied by the Collection of Algal Cultures, Institute of Plant Physiology, University of Göttingen, Germany.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

no data

Test temperature:

23+/-1ºC

pH:

Medium prepared using methanol stock solution: 8.3-8.9 over 3 days, 8.8-10.5 over 4 days.

Medium prepared using TBA stock solution: 8.4-8.7 over 3 days, 8.4-9.9 over 4 days.

Dissolved oxygen:

no data

Salinity:

not applicable

Nominal and measured concentrations:

Nominal concentrations in media prepared with methanol stock solution: 0(Control), 0.01, 0.032, 0.10, 0.32, 1.0, 3.2, 10.2 and 31.8 mg/L.
Nominal concentrations in media prepared with TBA stock solution: 0(Control), 0.009, 0.029, 0.094, 0.29, 0.94, 2.9, 9.4 and 29.5 mg/L.
It is likely that all exposure concentrations exceeded the solubility of the test substance in the test medium.

Details on test conditions:

TEST SYSTEM

- Test vessel: Conical flasks

- Type: closed with sponge cap

- Material, size, fill volume: glass, 200 mL containing 100 mL of test medium

- Aeration: no

- Initial cells density: 12000 cells/mL (methanol), 10000 cells/mL (TBA)

- Control end cells density: 2200000 cells/mL (methanol), 3000000 cells/mL (TBA)

- No. of vessels per concentration (replicates): 2

- No. of vessels per control (replicates): 2


GROWTH MEDIUM

- Standard medium used: yes except that the medium contained 150 mg/l of NaHCO3 instead of the 50 mg/l specified in the OECD guideline.

TEST MEDIUM / WATER PARAMETERS

- Source/preparation of dilution water: Milli-Q filtered water

- Culture medium different from test medium: no

- Intervals of water quality measurement: start and end of test


OTHER TEST CONDITIONS

- Sterile test conditions: yes

- Adjustment of pH: no

- Photoperiod: continuous

- Light intensity and quality: 60-120 μm/s/m2


EFFECT PARAMETERS MEASURED (with observation intervals if applicable): cell density at 0, 24, 48, 72 and 96 hours (approx)

- Determination of cell concentrations: Coulter Counter Model TAII


TEST CONCENTRATIONS

- Spacing factor for test concentrations: 3.2 approx

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 31.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: solvent: methanol

Remarks:

values calculated in retrospect from original cell numbers

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 29.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: solvent: TBA

Remarks:

values calculated in retrospect from original cell numbers

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

>= 31.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: solvent: methanol

Remarks:

values calculated in retrospect from original cell numbers

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

20.91 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: solvent: TBA

Remarks:

values calculated in retrospect from original cell numbers

Details on results:

- Exponential growth in the control (for algal test): yes

- Any stimulation of growth found in any treatment: some evidence of stimulation at concentrations >/=0.3 mg/L but it is not certain if it is significant.

- Effect concentrations exceeding solubility of substance in test medium: It is likely that all exposure concentrations exceeded the water solubility of the test substance i(0.0029 mg/L).

Reported statistics and error estimates:

The area under the growth curve (biomass) and growth rate parameters were calculated from the cell concentration data in accordance with the description of the test method.

Table 1. Cell concentrations [cells/mL] when methanol was used as a solvent

Conc. [mg/L]		10^4 cells/mL corrected for background
	Replicates	0 d	1 d	2 d	3 d	4 d
Control	1	1.5	2.0	11.9	79.2	231.2
	2	1	1.9	11.0	74.1	233.5
Solv. Control	1	1.2	2.1	10.5	76.7	205.9
	2	1	1.9	11.5	76.4	232.6
0.01	1	1.6	2.2	10.9	81.2	240.2
	2	1	2.0	12.7	81.2	233.5
0.032	1	1.4	2.1	11.1	76.8	244.1
	2	1	2.0	11.4	87.5	224.0
0.1	1	0.9	1.9	11.1	73.8	222.3
	2	0.8	2.2	12.3	95.4	286.6
0.32	1	1.1	1.9	11.5	77.1	218.3
	2	0.9	2.1	10.3	86.1	233.2
1	1	0.8	1.8	10.3	67.4	214.4
	2	0.9	2.1	9.4	69.1	219.9
3.2	1	0.9	2.3	9.8	57.8	199.4
	2	0.9	2.2	9.1	56.6	168.7
10.2	1	0.8	1.7	7.8	46.8	155.8
	2	1.1	1.7	9.1	44.5	131.8
31.8	1	0.8	9.1	6.4	45.3	170.1
	2	0.9	-0.9	2.3	49.5	173.5

 red value: was excluded from the calculation due to negative cell number

 

Table 2.Cell concentrations [cells/mL] when TBA was used as a solvent

Conc. [mg/L]		10^4 cells/mL corrected for background
	Replicates	0 d	1 d	2 d	3 d	4 d
Control	1	1	3.1	14.5	109.1	297.3
	2	1	3.1	13.2	97.5	291.6
Solv. control	1	1.1	3.1	14.4	108.5	319.4
	2	1	2.7	14.1	91.2	308.6
0.009	1	1	3.2	13.3	102.3	305.5
	2	1	2.6	14.9	103.1	277.1
0.029	1	1	3.1	12.6	101.2	317.8
	2	1	3.3	16.8	105.2	324.6
0.094	1	0.8	2.9	14.7	105.5	294.5
	2	0.9	3.3	16.3	115.1	316.1
0.29	1	1	2.7	15.2	104.7	259.7
	2	1	3.3	15.1	115.4	287.2
0.94	1	1	2.5	13.9	79.9	262.3
	2	1	3.1	14.4	107.9	329.6
2.9	1	1	2.8	12.9	83.8	287.1
	2	1	2.6	10.3	68.9	262.0
9.4	1	0.9	3.3	10.0	70.2	211.9
	2	0.9	3.2	10.1	60.5	192.5
29.5	1	1.1	2.6	8.7	62.1	176.3
	2	1.2	6.4	9.6	57.9	167.2

 

96 h -EC10 values mentioned in the report were 11 mg/L when TBA was used as a solvent and 7 mg/L when methanol was used as a solvent. No effects causing 50% inhibition of the growth rate were observed. The EC values were calculated by means of a parametric model developed by Kooijman et al. 1983.

Re-evaluation of data:

The 72 -h effect concentrations were calculated in retrospect according to the current OECD 201 (2011) guideline and the original cell numbers given in the report.

No differences were observed between the control and the solvent control. However since only duplicates were used, no statistical analysis could be performed. The solvent control was used as a worst case for the calculation of the growth rate inhibition rates.

1) Results on growth rate inhibition when methanol was used as a solvent:

Table 3: % Inhibition of growth rate at 72 h when methanol was used as a solvent, as calculated in retrospect according to the current OECD 201 standards using the orginal cell densities from the study report.

Nominal concentrations [mg/L]	% Growth Rate Inhibition at 72 h
0.01	1.94
0.032	0.18
0.1	-8.23
0.32	-3.78
1	-3.36
3.2	2.23
10.2	8.51
31.8	5.26

No growth rate inhibition up to 10% was observed after 72 h when methanol was used as a solvent resulting in an ErC10 (72 h) >= 31.8 mg/L.

2) Results on growth rate inhibition when TBA was used as a solvent:

Table 4: % Inhibition of growth rate at 72 h when TBA was used as a solvent, as calculated in retrospect according to the current OECD 201 standards using the original cell densities from the study report.

Nominal Concentrations [mg/L]	% Grwoth Rate Inhibition at 72 h
0.009	-1.75
0.029	-1.88
0.094	-6.89
0.29	-3.26
0.94	0.49
2.9	4.84
9.4	5.9
29.5	13.08

The EC10 value was calculated based on the dose-response curve. The ErC10 (72 h) was 20.914 mg/L (nominal) when TBA was used as a solvent.

No growth rate inhibition up to 50% was observed after 72 h of exposure when TBA was used as a solvent resulting in an ErC50 (72 h) > 29.5 mg/L (nominal).

Validity criteria fulfilled:

yes

Remarks:

The validity criteria outlined in the OECD guideline 201 version of 1984 are fulfilled

Conclusions:

The study resulted in an ErL50 (72 h) and ErL10 (72 h) of > 30.8 mg/L when methanol was used as a solvent. The ErL50 (72 h) and ErL10 (72 h) when TBA was used as a solvent were > 29.5 mg/L and 20.91 mg/L (nominal), respectively. The 72 h effect values were not presented in the report and therefore the calculations were made in retrospect based on the current OECD guideline 201 calculation criteria and the original cell numbers given in the study report.

Cross-reference 4

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 December 2011 - 15 December 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

The batch of Hexadecyltrimethoxysilane tested was a clear colourless liquid with a purity of 98.3% and not completely soluble in test medium at the loading rates initially prepared. The substance has a very low water solubility (prediction / substance: 6.5 E-4 mg/l, hydrolysis products: 1.5 mg/l), and degrades in contact with water (the predicted hydrolysis half-life is 4.6 h, information obtained by the sponsor).

The test substance was pipetted below the surface of the Milli-RO-water and stirred briefly to mix (5 minutes). Subsequently, synthetic sewage feed and sludge will be added (see section 6.4) and the test was initiated applying a 3 hour contact time. Optimal contact between the test substance and test organisms was ensured applying continuous aeration and stirring. The solution of the test substance in water was pH neutral.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

The sludge was coarsely sieved, washed and diluted with ISO-medium. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (3.0 g/l of sludge, as used for the test). The pH was 7.7 on the day of testing. The batch of sludge was used one day after collection; therefore 50 ml of synthetic sewage feed was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Post exposure observation period:

Oxygen consumption was measured and recorded for a period of 11 to 27 minutes after the 3 hours exposure period.

Test temperature:

Between 19.6 and 21.2°C

pH:

pH before additionof sludge: 7.2 - 7.4
pH after the 3-hour exposure periode: 8.0 - 8.2, except in the abiotic control (6.9)

Dissolved oxygen:

The oxygen concentration at the start of measurement was at least 6.9 mg O2/l

(Mean) respiration rates:
Control: 50 mg O2/l/h
10 mg/l: 48 mg O2/l/h
100 mg/l: 43 mg O2/l/h
1000 mg/l: 44 mg O2/l/h

Nominal and measured concentrations:

Nominal concentrations, three loading rates: 10, 100 and 1000 mg/l

Details on test conditions:

TEST SYSTEM
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: All glass
- Aeration:During exposure with clean, oil-free air
- No. of vessels per concentration (replicates): 10 mg/l (1), 100 mg/l (1), 1000 mg/l (3)
- No. of vessels per control (replicates): 2
- Biomass loading rate: 1.5 g/l suspended solids in final test mixture

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap-water purified by reverse osmosis (Milli-RO water).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The respiration rate from each vessel, in mg O2/l/hr, was calculated from the linear part of the respiration curve, which was generally between 2 and 7 mg O2/l.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

(loading rate)

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

(loading rate)

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

No statistically significant inhibition of the respiration rate of the sludge was recorded at a loading rate of 1000 mg Hexadecyltrimethoxysilane per litre (Two Sample t-Test: α=0.05 Toxstat).

There was no oxygen uptake from abiotic processes and the results at 1000 mg/l with a nitrification inhibitor showed a slightly higher heterotrophic inhibition of the respiration rate than the total inhibition of the respiration rate.

Results with reference substance (positive control):

The EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/l for total respiration (9.8 mg/l).

Reported statistics and error estimates:

ECx
For the reference substance the percentage inhibition was plotted against the logarithm of the concentrations and the EC50 was determined using linear regression analysis.

For Hexadecyltrimethoxysilane no EC50 could be calculated because the test substance proved to be non-toxic (EC50 > a loading rate of 1000 mg/l).

NOEC estimation
The NOEC was based on statistical analysis of the data. Data obtained for the test concentrations were compared with those obtained for the control using TOXSTAT Release 3.5, 1996, D.D. Gulley, A.M. Boelter, H.L. Bergman.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this present test Hexadecyltrimethoxysilane was not toxic to waste water (activated sludge) bacteria at or below a loading rate of 1000 mg/l (NOEC).

The EC50 exceeded a loading rate of 1000 mg/l.

Cross-reference 5

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1989-12-12 to 1989-12-16

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: 1.5 g of test substance was added to 1.5 L of dilution water and stirred vigorously for 24 hours on a magnetic stirrer at a temperature of 25+/1ºC. The aqueous phase was then decanted into the test vessel as the test medium.

- Evidence of undissolved material (e.g. precipitate, surface film, etc): It is likely that the solubility of the substance in the test medium was exceeded. An oil-like substance was observed on the surface of the test media.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM

- Common name: Zebrafish

- Source: obtained from a commercial hatchery; M.B. Ruysbroek B.V., Maassluis, The Netherlands.

- Length at study initiation (length definition, mean, range and SD): 2.2+/-0.1 cm

- Weight at study initiation (mean and range, SD): 0.08+/-0.01 g

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

215 mg/l as CaCO3

Test temperature:

25+/-1ºC

pH:

7.6-8.0

Dissolved oxygen:

6.1-8.5 mg/L

Salinity:

not applicable

Nominal and measured concentrations:

Nominal loading rates: 0(Control) and 1000 mg/L.

It is likely that the solubility of the substance in the test medium was exceeded. An oil-like substance was observed on the surface of the test media.

Details on test conditions:

TEST SYSTEM

- Test vessel: beaker

- Type (delete if not applicable): open

- Material, size, fill volume: glass, 2 L with 1.5 L fill volume

- Aeration: none

- Renewal rate of test solution (frequency/flow rate): daily

- No. of organisms per vessel: 10

- No. of vessels per concentration (replicates): 4

- No. of vessels per control (replicates): 4

- Biomass loading rate: 0.52 g/L

TEST MEDIUM / WATER PARAMETERS

- Source/preparation of dilution water: Synthetic freshwater (DSWL) prepared by the additional of salts to groundwater from near Linschoten, The Netherlands.

- Culture medium different from test medium: no

- Intervals of water quality measurement: Daily

OTHER TEST CONDITIONS

- Adjustment of pH: no

- Photoperiod: 16 h light, 8 h dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): mortality after 24, 48, 72 and 96 h.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: 0
- Mortality of control: 0
- Other adverse effects control: 0
- Abnormal responses: 0
- Effect concentrations exceeding solubility of substance in test medium: It is likely that the solubility of the substance in the test medium was exceeded. An oil-like substance was observed on the surface of the test media.

Reported statistics and error estimates:

No toxic effects were observed in the test and therefore statistical analysis of the results was not required.

Sublethal observations / clinical signs:

No toxic effects were observed in the test.

Validity criteria fulfilled:

yes

Conclusions:

A 96-h LL50 value of >1000 mg/L and NOELR of ≥1000 mg/L have been determined for the effects of the test substance on mortality of Brachydanio rerio based on nominal loading rate of the test substance.

Cross-reference 6

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002-04-15 to 2002-07-25

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Guideline study with restrictions. The study was conducted according to an appropriate OECD test guideline, with acceptable restrictions. The restrictions were that the analytical results showed that measured DOC concentrations were <5% of nominal. The results have been reinterpreted with reference to the measured concentrations of dissolved organic carbon in the test media

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

Test media were not prepared in accordance with OECD guidance for testing difficult substances

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Nominal treatments: 100 mg/L and 10 mg/L (10% concentration of 100 mg/L water-soluble fraction)
- Sampling method: A sample of test media were taken from all treatments at the start (before introducing the test organisms) and at the end of the test.
- Sample storage conditions before analysis: Not reported

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: A water-soluble fraction of the test substance was prepared by mixing 100 mg/l of the test substance with dilution water for 48 hours and then filtering (0.45 μm) to remove undissolved test substance. The water-soluble fraction was tested undiluted and at one-tenth of its original concentration.

- Controls: Dilution water

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM

- Strain: IRCHA

- Source: LPT Laboratory Culture

- Age at study initiation (mean and range, SD): 6-24 hours

ACCLIMATION

- Acclimation period: at least one week

- Acclimation conditions (same as test or not): yes

- Type and amount of food: algae (Scenedesmus subspicatus) plus a small quantity of sewage (only during the holding period) from the aerated phase of treatment

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

250 mg/L as CaCO3

Test temperature:

20+/-1ºC

pH:

7.61-8.15

Dissolved oxygen:

≥80% ASV

Nominal and measured concentrations:

Nominal loading rates: 0(Control), 100 mg/L and 10 mg/L (10% concentration of 100 mg/L water-soluble fraction).

A loading rate of 100 mg/L is above the expected solubility of the substance in the test medium. Measured dissolved organic carbon concentrations were below 5% of the theoretical values at the start and end of the test.

Details on test conditions:

TEST SYSTEM

- Test vessel:

- Type (delete if not applicable): open

- Material, size: glass, 38 mm diameter, 60 mm tall, 50 mL volume

- Aeration: none

- No. of organisms per vessel: 5

- No. of vessels per concentration (replicates): 4

- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS

- Source/preparation of dilution water: aerated reconstituted water

- Alkalinity: 0.8 mmol/L

- Ca/mg ratio: 4:1

- Culture medium different from test medium: no

- Intervals of water quality measurement: Start and end ot test

OTHER TEST CONDITIONS

- Adjustment of pH: no

- Photoperiod: 16 h light/8 h dark

- Light intensity: 500 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Mobility after 24 and 48 hours

TEST CONCENTRATIONS

- Spacing factor for test concentrations: 10

Reference substance (positive control):

no

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

< 10 other: percent concentration of the 100 mg/l water-soluble fraction

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Immobilization of control: 5%

- Effect concentrations exceeding solubility of substance in test medium: A loading rate of 100 mg/L is above the expected solubility of the substance in the test medium.

Reported statistics and error estimates:

There were insufficient toxic effects to determine an EL50 value. The EC50 and NOELR values were determined directly from the raw data.

Table 1. Test results

Nominal test substance loading rate (mg/L)	Mean percentage immobilisation after 24 hours	Mean percentage immobilisation after 48 hours
0 (Control)	0	5
10 (10% concentration of 100 mg/L water-soluble fraction)	0	10
100	0	15

Table 2. Results of analysis of test media

Nominal test substance loading rate (mg/L)	Theoretical DOC concentration (mg/L)	Actual DOC concentration at start of test (mg/L)	Actual DOC concentration at end of test (mg/L)
10 (10% concentration of 100 mg/L water-soluble fraction)	6.6	<0.5	0.5
100	65.8	<0.5	0.9

Validity criteria fulfilled:

yes

Conclusions:

A 48-h EL50 value of >100 mg/L has been determined for the effects of the test substance on mobility of Daphnia magna based on nominal loading rate of the test substance. The NOELR in the test corresponded to a <10% concentration of the 100 mg/L water soluble fraction.

Cross-reference 7

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Feb - 08 Mar 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Staatliches Gewerbeaufsichtsamt Hildesheim, Germany

Analytical monitoring:

yes

Remarks:

LC-MS/MS and TOC

Details on sampling:

- Concentrations: Control, 10 mg/L (nominal)
- Sampling for determination of the test item: at least once within 7 d in fresh media at the start of an exposure interval and in old media at the end of an exposure interval (48 or 72 h). Furthermore once during the test, samples of the saturated solution and the control taken during the preparation phase of the saturated solution and during the exposure phase (i.e. at 24, 20, 0 and 48 h) were analyzed via LC-MS/MS and also via TOC-analysis to demonstrate the establishment of an equilibrium of the test item concentration.
- Sampling for the analytical monitoring: during the preparation of the saturated solution and at the start of an exposure interval, samples of the fresh media were taken at 24 h (after addition of the test item and a few minutes of stirring), at 20 h (approx. 4 h after the start of the stirring period during the preparation of the saturated solution) and at 0 h (after preparation of the saturated solution). At the end of an exposure interval (48 or 72 h), samples for analyses were taken from the test vessels. For analysis of the TOC, separate replicates of the saturated solution and the control were prepared without daphnids and food algae and analyzed. For the longest exposure interval of 72 h, samples were taken at the start (0 h) and at the end of the exposure interval (72 h) once within the test period.
- Sample storage conditions before analysis: all samples were stored at room temperature until sample preparation and in an autosampler until start of analysis, if necessary

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: the solution was prepared with a nominal concentration of 10 mg/L in Elendt M4 medium one day prior to the start of each exposure interval. 22.48 µL test item were slowly added by pipette to 2L Elendt M4 medium. The mixture was stirred slowly for 24 h with a magnetic stirrer at room temperature. Subsequently the solution was allowed to stand for 1 h for separation of the phases and then the saturated solution was removed from the center of the aqueous phase. Once during the test the establishment of an equilibrium of the test item concentration was shown by analysis during the preparation phase (0 and 4 h after stirring) and during the exposure phase (0 and 48 h).

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain/clone: clone 5
- Source: daphnids were bred at the test facility (origin: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany)
- Culturing conditions: in glass vessels (2 - 3 L) with approx. 1.8 L medium (Elendt M4), at approx. 20 °C, in an incubator, 16 h illumination; light intensity of max. 1500 Lux. Daphnids were fed at least 5 times per week ad libitum with a mix of unicellular green algae with an algae cell density of > 10^6 cells/mL
- Age of daphnids at test start: < 24 h. Juvenile daphnids were removed from the culture vessels at the latest 24 h before the start of the exposure and discarded. The juveniles born within this period of max. 24 h preceding the exposure were used for the test. No first brood progeny was used for the test.
- Feeding during test: yes
- Food type and amount: Pseudokirchneriella subcapitata (1.06 - 1.40 mL) and Desmodesmus subspicatus (0.623 -0.786 mL) suspension was provided as food corresponding to 0.2 mg C per Daphnia and day. There was variation according to the density of the algae suspension, but it was the same for all test groups on each feeding day.
- Frequency: daily

ACCLIMATION
- Acclimation period: non necessary since culture medium same as test medium

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Hardness:

158 - 172 mg CaCO3/L

Test temperature:

19.8 - 20.4 °C (water temperature)

pH:

7.06 - 8.45

Dissolved oxygen:

7.63 - 9.64 mg/L

Nominal and measured concentrations:

Nominal loading rate: 10 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL glass beakers
- Type (delete if not applicable): beakers were loosely covered with watch glasses
- Fill volume: 50 mL
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): three times per week
- No. of organisms per vessel: 10 (1 daphnid per replicate was used in the control)
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4, according to OECD 211
- Culture medium different from test medium: no
- Intervals of water quality measurement: once within 7 d, in fresh media at the start of an exposure interval (0 h) and in old media at the end of an exposure interval (48 or 72 h), in one replicate of the control and the saturated solution. The water quality parameters in fresh media were measured in an additional replicate without daphnids of the saturated solution and the control. At the end of an exposure interval, the water quality parameters of the old media were measured in a test vessel of the saturated solution and the control, which contained daphnids and food algae. The temperature in the incubator was recorded throughout the test.

OTHER TEST CONDITIONS
- Photoperiod: 16/8 h light/dark cycle
- Light intensity: 1500 Lux

EFFECT PARAMETERS MEASURED: immobile daphnids were observed and recorded once per day and offspring was counted. Dead specimens were removed. The neonates were removed after counting and before addition of algae. The number of aborted eggs or dead offspring was recorded. Abnormalities (e.g. swimming behavior, number of males and winter eggs) were observed and recorded daily. At the end of the test, the total length excluding the anal spine of each survived parental daphnid and the mean dry weight of the survived parental daphnids of the saturated solution and the control were determined (these information were not used for determination of a NOEC). The time to first brood, the intrinsic rate of population increase and the number and size of first brood per animal were reported, but not used for endpoint calculations.

TEST CONCENTRATIONS
- Range finding study: yes (static conditions, 48 h, 2 replicates)
- Test concentrations: control, 2 mg/L
- Results used to determine the conditions for the definitive study: no mortalities were observed in the range finding test

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

>= 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

- Observations on body length and weight: the mean values of the body length (excluding the anal spine) of the parental daphnids in the saturated solution were 4.20 mm per daphnid and 4.50 mm per daphnid in the control group. The mean values of the dry weight of the parental daphnids were 0.80 mg per daphnid in the saturated solution and 0.64 mg per daphnid in the control.
- Other biological observations: stillborn juveniles and aborted eggs produced by the parental daphnids after 21 days was 0. No males were observed in the control or in the test groups during the test. No ephippia were observed in the control or in the test groups during the test.
- Mortality of control: no

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 (24 h) = 2 mg/L (95% CI: 1.8 - 2.28 mg/L)

Reported statistics and error estimates:

Since no mortality appeared in this study, statistical evaluation for the adult mortality was not necessary. Further information on statistics can be found in the field any other information on results incl. tables.

ANALYTICAL RESULTS

The measured concentrations of the test item determined via LC-MS/MS during the preparation phase of the saturated solution and during the exposure were all below the LOQ (152 µg/L) of the analytical method. The measured TOC concentrations were around the LOQ of 2 mg C/L in all analyzed samples. This indicates that the test item is hardly soluble as well as completely hydrolyzed during the exposure. Since the test item concentrations could not be quantified, the nominal concentration of the test item was used for the evaluation of the NOEC and LOEC.

Table 1: Measured TOC Concentrations during the Definitive Test

Sampling date	Day 0 Fresh media (0 h)	Day 2 Old media (48 h)	Day 9 Fresh media (0 h)	Day 12 Old media (72 h)	Day 13 Preparation of the saturated solution (0 h)	Day 13 Preparation of the saturated solution (after 4 h stirring)	Day 14 Fresh media (0 h)	Day 16 Old media (48 h)
Nominal test item concentration [mg/L]	Total Organic Carbon (TOC)
	Measured concentration [mgC/L]
10.0 (saturated solution)	2.70	< 2.00	< 2.00	< 2.00	< 2.00	3.65	< 2.00	< 2.00
Control	2.66	< 2.00	3.02	< 2.00	< 2.00	< 2.00	< 2.00	< 2.00

BIOLOGICAL RESULTS

Table 2: Effects on Reproduction for all parental Daphnids

Nominal test item concentration	Mean number of offspring per survived / introduced parental daphnid
[mg/L]	Mean	SD	CV
10.0	82.7	9.89	12.0
Control	93.7	13.6	14.5

Table 3: First Appearance of Living Juveniles and Mean Number of Broods in the Individual Groups

Nominal test item concentration [mg/L]	Day of first appearance of living juveniles at the parental daphnid in replicate no.										First
											appearance
	1	2	3	4	5	6	7	8	9	10	mean day
10.0 (saturated solution)	8	8	8	9	8	9	9	9	8	8	8.4
Control	9	9	8	9	9	9	9	9	8	8	8.7

STATISTICAL ANALYSIS

No statistically significant difference of the reproductive output in comparison to the reproductive output in the control was determined at the saturated solution.

Adult mortality was not observed in this study. Therefore, no statistical evaluation was carried out for this parameter.

Number of living juveniles per survived parental Daphnid:

- Shapiro-Wilk's test on normal distribution: normality check was passed (p > 0.01).

- Levene's Test on variance homogeneity (with Residuals): the Levene test indicates variance homogeneity (p <= 0.010). Variance homogeneity check was passed (p > 0.01). Normal-distribution and variance-homogeneity requirements are fulfilled.

- Two-sample Welch-t-test Procedure (two sided test) with cumulative offspring per survived parent at 21 d: two-sample comparison of treatments with "Control". Significance was Alpha = 0.050, two-sided; Mean: arithmetic mean; n: sample size; s: standard deviation; MDD: minimum detectable difference to Control (in percent of Control); t: sample t; p(t): probability of sample t for ; the differences are significant in case p(t) <= Alpha (Control(c) and treatment(t) variance was applied: s^2(c)/nc + s^2(t)/nt, each).

Table 4: Two-sample Welch-t-test Procedure

Treatm. [mg/L]	mean	s	df	%MDD	t	t*	sign.
control	93.7	13.58
10	82.7	9.89	16	12	-2.07	0.55	non-sign.

VALIDITY CRITERIA

Table 5: Validity criteria

Criterion from the guideline	Outcome	Validity criterion fulfilled
The mortality of the parent animals (female Daphnia) does not exceed 20% at the end of the test.	% mortality of the Adult Daphnids after 7, 14 and 21 days of exposure was 0.	yes
The mean number of living offspring produced per parent animal surviving at the end of the test is ≥ 60.	The average number of living juveniles at the end of the test after 21 days per survived parental daphnid was 93.7 in the control group	yes

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables".

Conclusions:

The study conducted according to the OECD guideline 211 and GLP did not show any long term effects to the test organism Daphnia magna resulting in a NOELR (21 d) >= 10 mg/L (nominal).

Cross-reference 8

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Name:

Harmonised Reconsile Consortia C&L according to CLP Regulation

Not classified

Implementation:

EU

Type of classification:

self-classification

Related composition:

Hexadecyltrimethoxysilane

Reason for no classification:

data conclusive but not sufficient for classification

Reason for no classification:

data conclusive but not sufficient for classification

Reason for no classification:

data conclusive but not sufficient for classification

Reason for no classification:

hazard class not assessed

Reason for no classification:

data conclusive but not sufficient for classification

Reason for no classification:

data conclusive but not sufficient for classification

Reason for no classification:

data conclusive but not sufficient for classification

Reason for no classification:

data conclusive but not sufficient for classification

Reason for no classification:

data conclusive but not sufficient for classification

Reason for no classification:

data conclusive but not sufficient for classification

Reason for no classification:

data conclusive but not sufficient for classification

Reason for no classification:

data conclusive but not sufficient for classification

Reason for no classification:

data conclusive but not sufficient for classification

Reason for no classification:

data conclusive but not sufficient for classification

Reason for no classification:

data conclusive but not sufficient for classification

Reason for no classification:

data conclusive but not sufficient for classification

Reason for no classification:

data conclusive but not sufficient for classification

Reason for no classification:

data conclusive but not sufficient for classification

Reason for no classification:

data conclusive but not sufficient for classification

Reason for no classification:

data conclusive but not sufficient for classification

Reason for no classification:

data conclusive but not sufficient for classification

Reason for no classification:

data conclusive but not sufficient for classification

Reason for no classification:

data conclusive but not sufficient for classification

Reason for no classification:

data lacking

Reason for no classification:

data conclusive but not sufficient for classification

Reason for no classification:

data conclusive but not sufficient for classification

Reason for no classification:

data conclusive but not sufficient for classification

Reason for no classification:

data lacking

Reason for no classification:

data conclusive but not sufficient for classification

Reason for no classification:

data lacking

Reason for no classification:

data conclusive but not sufficient for classification

Reason for no classification:

data conclusive but not sufficient for classification

Reason for no classification:

data conclusive but not sufficient for classification

Reason for no classification:

data conclusive but not sufficient for classification

Reason for no classification:

data conclusive but not sufficient for classification

Signal word:

No signal word

Cross-reference 9

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

Version / remarks:

Published method: NMR-Spectroscopic Investigations on the Hydrolysis of Functional Trialkoxysilanes, M. Brand, A. Frings, P. Jenkner, R. Lehnert, H. J. Metternich, J. Monkiewcz, J. Schram, Verlag der Zeitschrift für Naturforschung Tübingen 54b, 155-64, (1999)

Deviations:

yes

Remarks:

Due to the low solubility of the substance, the water/solvent ratio, was adjusted. Furthermore since the hydrolysis speed of the substance is low, the duration of the studies and the measurement intervals were prolonged to 14 d and daily measurement.

Principles of method if other than guideline:

- Principle of test: Three different series of measurements were carried out at pH 7, pH 10 and pH 4 according to the published method “NMR-Spectroscopic Investigations on the Hydrolysis of Functional Trialkoxysilanes, M. Brand, A. Frings, P. Jenkner, R. Lehnert, H. J. Metternich, J. Monkiewcz, J. Schram, Verlag der Zeitschrift für Naturforschung Tübingen 54b, 155-64, (1999)”.

GLP compliance:

no

Remarks:

The laboratory is accredited from the German Akkreditation Body according to DIN EN ISO/IEC 17025

Radiolabelling:

no

Analytical monitoring:

yes

Remarks:

1H-NMR spectra

Details on sampling:

- Sampling intervals for the parent/transformation products: At the start and then on a daily basis over two weeks.
- Sampling method: All tests were done directly within the NMR tubes.

Details on test conditions:

TEST SYSTEM
- Type, material and volume of test flasks, other equipment used: NMR tubes
- Is there any indication of the test material adsorbing to the walls of the test apparatus: Yes, therefore all NMR tubes were pretreated with Hexamethyldisilazane to prevent interactions with the glass surface, rinsed with water and dried overnight in a drying cabinet.

TEST PREPARATION
pH 7: 0.50 mL acetone-d6 and 0.10 mL H2O were transferred to a NMR tube and mixed. Using a syringe 10 µl of Hexadecyltrimethoxysilane were added (the resulting concentration of silane is 15 g/L or 0.043 mol/L).
pH 10: 0.50 mL acetone-d6 and 0.10 mL H2O were transferred to a NMR tube and mixed. The solution was adjusted to pH 10 with NaOH. Using a syringe 10 µl of Hexadecyltrimethoxy-silane were added (the resulting concentration of silane is 15 g/L or 0.043 mol/L).
pH 4: 0.50 mL acetone-d6 and 0.10 mL H2O were transferred to a NMR tube and mixed. The solution was adjusted to pH 4 with HCl. Using a syringe 10 µl of Hexadecyltrimethoxysilane were added (the resulting concentration of silane is 15 g/L or 0.043 mol/L).

OTHER TEST CONDITIONS
- Adjustment of pH: with NaOH or HCl

Duration:

14 d

pH:

4

Initial conc. measured:

94.2 other: mol%

Duration:

14 d

pH:

7

Initial conc. measured:

100 other: mol%

Duration:

14 d

pH:

10

Initial conc. measured:

98.7 other: mol%

Number of replicates:

Not reported

Positive controls:

not specified

Negative controls:

not specified

Preliminary study:

yes (feasibility study), see field "any other information on material and methods incl. tables"

Transformation products:

yes

No.:

#1

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

- Pathways for transformation: The three SiOMe-groups of the silane will react with water and deliberate methanol. The SiOH-groups are not stable and generate Si-O-Si bonding and crosslink eventually.

% Recovery:

59.9

pH:

4

Duration:

14 d

% Recovery:

84.4

pH:

7

Duration:

14 d

% Recovery:

82

pH:

10

Duration:

14 d

pH:

7

DT50:

> 14 d

Type:

not specified

pH:

4

DT50:

> 14 d

Type:

not specified

pH:

10

DT50:

> 14 d

Type:

not specified

Hydrolysis at pH 4:

After 14 days 60 mol% of the SiOCH₃groups remained intact and 40 mol% were hydrolyzed:

Hydrolysis at pH 7:

After 14 days 94.4 mol% of the SiOCH₃groups remained intact and only 5.6 mol% were hydrolysed.

Hydrolysis at pH 10:

After 14 days 82 mol% of the SiOCH₃groups remained intact and 18 mol% were hydrolysed.

Table 1: Hydrolysis speed at different pH values over time.

	% Hydrolysis (= mol % methanol)
Time [d]	pH 7	pH 10	pH 4
0	0	1.3	5.8
1	0,6	4.6	7.9
2	1	5.4	10.7
3	1.2	7.8	13.2
4	1.6	-	17.6
5	-	-	21.8
6	-	10.5	23.9
7	2.5	11.8	26.6
8	2.8	13.3	28.9
9	3.2	13.8	31.5
10	3.7	14.5	32.4
11	4.3	-	35
12	-	-	37.4
13	-	17.7	39.1
14	5.6	18	40.1

Validity criteria fulfilled:

not applicable

Cross-reference 10

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 Mar - 04 Aug 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 7-8 weeks
- Weight at study initiation: males: 155 – 192 g; females: 113 – 163 g
- Fasting period before study: Not reported
- Housing: Housed in groups of 5 animals / sex / group / cage in IVC cages (type IV, polysulphone cages) on Altromin saw fibre bedding
- Diet: Altromin 1324 maintenance diet for rats and mice available ad libitum
- Water: Tap water sulphur acidified to a pH of approximately 2.8 available ad libitum
- Acclimation period: At least 5 days

DETAILS OF FOOD AND WATER QUALITY:
Drinking water: municipal residue control, microbiological controls at regular intervals;
Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins BioPharma Product Testing Munich GmbH. There were no known contaminants in the feed or water at levels that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Details on route of administration:

The test item formulation and vehicle were administered as a single daily dose to the animals by oral gavage. The application volume for all groups was 4 mL/kg body weight.

Vehicle:

corn oil

Remarks:

dried and de-acidified

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was weighed into a tared plastic vial and the vehicle was added to give the appropriate final concentration of the test item (w/w). The formulation was vortexed and/or stirred until visual homogeneity was achieved. After homogenization the formulation was overlaid with argon to prevent instability caused by repeated contact of the test item formulation with air. Formulates were kept under magnetic stirring during the daily administration.
Based on the results of stability testing, the test item formulations were prepared at least every 15 days. The prepared formulation was stored protected from light and at room temperature.


VEHICLE
- Justification for use and choice of vehicle: The vehicle was selected in consultation with the sponsor based on the test item’s characteristics.
The corn oil was dried and de-acidified as follows: corn oil was filtered through a mixture of activated silica gel 60 and aluminium oxide (1:1, volume/volume), which has been filled into a glass chromatography column to three quarters of its height.
For filtering, a vacuum of 75 mbar was applied. The dried and de-acidified vehicle was overlaid with argon and stored until usage.
- Concentration in vehicle: 25, 75 and 250 mg/mL
- Amount of vehicle: 4 mg/mL
- Lot/batch no.: MKCK6411

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before beginning of the treatment period, formulation samples were prepared and analyzed in order to obtain knowledge about stability and homogeneity of the test item in the selected vehicle as part of a separate GLP study.
Prestart homogeneity investigation was included on the samples collected from various levels (top, middle and bottom) at high- and low-dose concentrations.
As the test item was shown to be homogenous according to the separate GLP study (after 30 min without stirring), samples were not collected during the study for the investigation of homogeneity and only samples were taken for substance concentration verification in study weeks 1, 5, 9 and in the last week of treatment (16 samples in total). The mean recoveries observed were between 97.4% and 104.7% of the nominal value for the low-dose dose group, between 97.7% and 102.8% of the nominal value for the mid-dose group and between 91.5% and 101.5% of the nominal value for the high-dose group. The mean overall recoveries observed in the low-, mid- and high dose groups were 100.4%, 100.4%, and 97.4% of the nominal concentration, respectively.
Nominal concentrations were confirmed for all dose groups, as measured concentrations were within the acceptance criterion of 10%.

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily, 7 days/week

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 (treatment and control groups)
5 (recovery group for control and high-dose)

20 additional animals (10 males and 10 females) will be used in satellite recovery groups for assessment of possible delayed toxic effects (5 male and 5 female animals per group).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: I n a previous dose range finding study in which 3 animals / sex / dose were dosed at 100, 300 and 1000 mg/kg bw/day, respectively, for 14 consecutive days test item-related effects on body weight development of mid- and high-dose male animals and food consumption of male and female mid and high-dose animals were observed.
- Rationale for animal assignment: Before the first administration all animals used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females, respectively (randomisation was performed with Microsoft Excel 2010 software).
- Fasting period before blood sampling for clinical biochemistry: yes; overnight
- Rationale for selecting satellite groups: In order to allow a detection of possible delayed occurrence or persistence of, or recovery from toxic effects, the animals in the recovery groups are observed for a period of 28 days following the last administration.
- Post-exposure recovery period in satellite groups: 28 days

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations are made once daily.
The health condition of the animals was recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first exposure and at least once a week thereafter.
- Detailed cage side observations (outside the home cage in a standard arena) included: spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment and recovery period.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Weekly during the treatment and recovery periods

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmological examinations, using an ophthalmoscope were made on all animals before the first administration and in the last week of the treatment period. At the end of the recovery period the ophthalmological examinations were not recorded for the recovery animals.
- Dose groups that were examined: All groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment and recovery period as part of the sacrifice of the animals
- Anaesthetic used for blood collection: Yes; ketamine/xylazin
- Animals fasted: Yes; overnight
- How many animals: All surviving animals of the treatment period and 4 weeks after the last administration all surviving animals of the recovery period (study day 119)
- Parameters checked in tables 1 and 2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment and recovery period as part of the sacrifice of the animals
- Animals fasted: Yes; overnight
- How many animals: All surviving animals of the treatment period and 4 weeks after the last administration all surviving animals of the recovery period (study day 119)
- Parameters checked in table 3 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Urinalysis performed on all animals as part of the scrifice of the animals
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters checked in table 5 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once before the first exposure and towards the end of the exposure period but not earlier than in week 11. In the last week of the recovery period the behavioural observations were not performed for the recovery animals.
- Dose groups that were examined: All dose groups
- Battery of functions tested:

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. One day after the last administration (study day 91) all animals of the treatment period and 4 weeks after the last administration all animals of the recovery period (study day 119) were sacrificed using anesthesia (ketamine, xylazin) followed by exsanguination and were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.
Vaginal smears were examined on the day of necropsy to determine the stage of oestrous cycle.
The wet weight of the organs presented in Table 6 were taken from sacrificed animals as soon as possible. Paired organs were weighed together. Weight of thyroid/parathyroid glands was measured after fixation.
The tissues presented in Table 7 from all animals were preserved in 4% neutral-buffered formaldehyde except eyes, testes and epididymides which were fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 70% ethanol.

HISTOPATHOLOGY: Yes; organs presented in Table 7 were examined histopathologically after preparation of paraffin sections and haematoxylin-eosin staining for the animals of the control and high-dose groups sacrificed at the end of the treatment period.
For testis, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.
The examinations mentioned above were not extended to animals of all other dosage groups as there were no treatment-related changes observed in the high-dose group.
Any gross lesion macroscopically identified was examined microscopically in all animals. Discoloration possibly due to the test item was evaluated in the organs of all dose groups.

Other examinations:

T3, T4, TSH: For an evaluation of test item-related effects on the pituitary-thyroid axis and thyroid hormones, serum samples of all animals was retained at the end of treatment (80 animals) and recovery (20 animals) and stored at < -15 °C. T3, T4 and TSH serum levels were determined of main study animals (80 animals; Table 4).

Examination of fertility parameters:
Daily over a period of 8 days, the oestrous cycle of all female animals was monitored in the last week of treatment. In the recovery animals the oestrous cycle was also monitored during the last week of the recovery period.
At necropsy (one day after the last administration) and at the end of the recovery period, left epididymis, left testis and left vas deferens were separated and used for evaluation of sperm parameters.
Epididymal sperm motility and testicular sperm count were evaluated in all male animals using Hamilton Thorne Sperm Analyser. Sperm morphology slides were prepared and evaluated from all male animals (main and recovery).

Statistics:

A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights was performed for each gender by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. Furthermore, statistical comparisons of data acquired during the recovery period may be performed with a Student’s t-Test or Mann-Whitney U-Test when appropriate. These statistics were performed with Ascentos 1.3.4 software or GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Slight to severe salivation was noted in 4/10 males and 3/10 females of the mid-dose group, 5/15 males and 6/15 females of the high-dose group. Furthermore, moving the bedding was noted in 2/10 males and in 5/10 females in the mid-dose group and 8/15 males and 13/15 females in the high-dose group. Slight salivation was recorded for 1/15 females in the control group on one single day. The clinical signs of moving the bedding and salivation in the mid- and high-dose groups were observed immediately after the dose administration during the treatment period and were therefore considered to be signs of discomfort, rather than a systemic adverse effect of the test item. These signs were still observed on the first day of the recovery period in the male and female high-dose group.
Further observations in the high-dose group were hunched posture in 3/15 males and 3/15 females, piloerection in 7/15 males and 4/15 females. 1/15 Male high-dose animals showed abnormal breathing. These findings were observed on several or single days in most of the animals. As no such findings were observed in other dose groups a treatment-related effect of the test item is considered.
Clinical symptoms like hairless area and scratch/cut were observed mostly transiently or on single days in animals of the control, low-, mid- or high-dose group in males or females and are were within the normal background frequency without toxicological relevance.
On the first day of the recovery phase moving bedding was noted in 3/5 high-dose males and 4/5 high-dose females, piloerection in 1/5 high-dose males and 2/5 high-dose females, slight salivation on two days in 1/5 high-dose females and hunched posture in 1/5 high-dose females. Hairless area was seen on one day in 1/5 female control animals. These findings were seen at the beginning of the recovery phase and therefore considered to be incidental and without toxicological relevance.

No statistically significant effects were noted on detailed clinical examination parameters during the treatment and recovery period when compared to the control group.

For a detailed desription of (detailed) clinical observations in tabular form please refer to the attachment.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred in the control or any of the dose groups during the treatment period of this study and the recovery period. All male and female animals survived to the scheduled necropsy.
For a detailed desription of the findings in tabular form please refer to the attachment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Treatment with the test item had a toxicological relevant effect on the body weight development in high-dose males and females and mid-dose males during the treatment period.
A statistically significant decrease of body weight was found in high-dose males on day 8 (4.64% below control) and continued during the entire treatment period in the high- and mid-dose group (high-dose group: up to 20.94% below control, mid-dose group: up to 15.63% below control). In high-dose females, statistically significant decreases were found on study days 29 and 36 and additionally, from day 57 until the end of the treatment period (up to 8.88% below control). Furthermore, the weekly body weight change, was seen regularly during the treatment period with statistical significant decrease in the male mid- and high-dose group and in single weeks in the female mid- and high-dose groups. One single statistical significance was found in the first week of the treatment period in the female low-dose group (7.8 g in the low-dose group, 13.20 g in control). Overall, the body weight change between day 1 to day 90 was statistically significantly decreased amounting 132.5 g in the male mid-dose and 104.20 g in the male high-dose group when compared to the control with 200.40 g. The statistically significant decrease in body weight change in the female high-dose group was 55.07 g compared to 73.93 g in the control group. In the control and all dose groups mean body weight was increased on treatment day 90 when compared to day 1.
During the entire recovery period the mean body weight was statistically significantly decreased in the male and female high-dose group when compared to the respective control groups, but the mean body weight change increased with statistical significance in the first two weeks in the male high-dose group and in the first week in the female high-dose group. A statistical significant decrease in body weight change was noted in week 3 of the recovery period in the female high-dose group when compared to control. Overall, the body weight change showed an increase on day 118 compared to day 90 in males and females with statistical significance in the male high-dose group (38.80 g compared to 18.60g in the control).
For a detailed desription of the body weight and body weight change in tabular form please refer to the attachment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The measurement of food consumption showed effects in the male and female high-dose group, which were considered to be related to the treatment with test item.
Decreased food consumption in the high-dose male group was found with statistically significant differences compared to the control group in several weeks during the treatment period amounting up to 23.15% below control. Additionally, reduced food consumption with statistical significance was found in the female high-dose group in most weeks of the treatment period (up to 20.05% below control).
The food consumption during the recovery phase was comparable to the respective control group in the male and female high-dose group.
For a detailed desription of the food consumption in tabular form please refer to the attachment.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not specified

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The test item had no toxicologically relevant effects on haematological and coagulation parameters of male and female animals analysed at the end of the treatment and recovery period of this study.
At the end of the treatment period, the measurement of WBC showed an increase of the group mean value in the low-dose and the high-dose group in males compared to the control, but only for mid- and high-dose in females. The WBC mean value was statistically significantly increased in the male high-dose group (75.75% above control) and in the female mid-dose (80.99% above control) and high-dose group (140.52% above control). At the end of the recovery period, no such increase was found in high-dose males and females. The group mean values for WBC were 11.84% below control in males and statistically significantly 56.45% below control in females. In the absence of specific findings in differential blood cell count and no test item-related abnormalities during histopathological evaluation, the statistical significances in WBC count are assumed to be of no toxicological relevance.
The red blood cell parameters RBC, HGB and HCT were slightly and statistically significantly increased in the high-dose male group (7.95%, 6.50% and 7.95% above control) whereas in females, a slight statistically significant increase was seen in the mid-dose group for RBC (6.62% above control) and for HGB and HCT in the mid- and high-dose group (mid-dose group: 6.84% and 6.28% above control, high-dose group: 5.24% and 5.67% above control). The changes were slight in both genders and seen without dose dependency in the female groups. Furthermore, histopathological evaluation showed no test item-related findings. Therefore, no toxicological relevant effect is concluded for red blood cell parameters. The single statistically significant finding in differential blood cell count in mid-dose males for neutrophils (36.76% above control) is considered to be incidental.
At the end of the recovery period, statistical significances from the respective control group were noted in high-dose males for RBC, MCH, MCHC, RET, NEUT and LUC and in females for HCT, LYM, NEUT and BASO. As the statistically significant differences were only slight or were not observed at the end of the treatment period, no toxicological relevance is concluded.
The coagulation parameter PT, was found with a slight statistically significant increase in the mid-dose (5.30% above control) and high-dose female group (6.71% above control) at the end of the treatment period and is assumed to be of no toxicological relevance as no statistical significant increase for PT was seen in the male dose groups. No statistical significant changes were observed for any parameter of the coagulation parameters for the male and female high-dose group at the end of the recovery period.
For a detailed desription of the findings in tabular form please refer to the attachment.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The test item is considered to have no toxicologically relevant effect on clinical biochemical parameters and hormone analysis of male and female animals analysed at the end of the treatment and recovery period of this study.
At the end of the treatment period, a statistically significant increase of the parameters TBIL (36.07% above control), CHOL (24.67% above control) and LDL (99.53% above control) was found in the male high-dose group and for LDL additionally in the male mid-dose group (57.85% above control).
A statistically significant increase of TBIL was also seen in high-dose females (31.55% above control), but not for CHOL. The slight increase for CHOL between the female dose groups when compared to the control group was not statistically significant. An increase of 140.07% above control was found at the end of treatment in high-dose females for TBA, whereas the increase in the high-dose male group was found without statistical significance. In the absence of toxicological relevant findings of liver specific parameters at clinical pathological evaluation and, additionally, the histopathological evaluation, which showed no test item-related findings, no toxicological relevance is assumed for the statistically significant changes in males or females at the end of the treatment period.
In females, the parameters TP and ALB showed a statistically significant decrease in the high-dose group when compared to control (6.34% and 5.52% below control) without dose dependency and were, therefore, considered to be without toxicological relevance.
The statistically significant decrease of 32.61% below control for ALAT in the male low-dose group and Crea (17.83% below control) in the female mid-dose group are considered to be incidental.
At the end of the recovery period, a statistical significance was only found for ALAT in the high-dose female group (36.11% below control).
There were no statistically significant changes in hormone analysis (T3, T4, TSH) at the end of the treatment and recovery period. Mean values of all male and female dose groups were comparable to the control group and no toxicological effect of the test item is concluded.
For a detailed desription of the findings in tabular form please refer to the attachment.

Endocrine findings:

not specified

Description (incidence and severity):

The following ED-related parameters were investigated in the study: T3, T4 and TSH; histopathology of epididymides, mammay gland, ovary, oviduct, prostate (with seminal vesicles and coagulation gland), testes, thyroid, uterus with cervix, vagina, adrenals and pituitary; organ weights of epididymides, liver, ovary, prostate, seminal vesicles, testes, thyroid, uterus, adrenals, brain and pituitary; estrous cyclicity; sperm motility, testicular sperm count and sperm morphology. For details, please refer to the respective result fields and the endpoint summary.

Urinalysis findings:

no effects observed

Description (incidence and severity):

The urinalysis of animals sacrificed at the end of the treatment and recovery period revealed no toxicological relevant effects and all urinary parameters were in the normal range of variation. No marked differences with toxicological relevance were noted between the dose groups and compared to the control groups.
For a detailed desription of the findings in tabular form please refer to the attachment.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

In both males and females, no toxicological effects were observed in any of the parameters of the functional observational battery before and at the end of the treatment when compared to controls.
Before the start of the treatment period (week -1) statistically significant differences were observed for animal sleeping (50% below control), moving in the cage and grooming in the male low-dose group and in females for animal sleeping (100% below control), moving in the cage (170% above control) in the mid-dose group.
In the last week of the treatment period (week 13), statistical significance was found for animals sleeping (50% above control) and moving in the cage (100% below control) in all male dose groups, for twitches in the low-dose male group and a decrease in defaecation in all male dose groups (low-dose: 86.36%, mid-dose: 72.73%, high-dose: 90.91% below control). No statistically significant differences to the control group were observed for all female dose groups.
The single statistically significant differences were noted before start of the first treatment or were observed in one gender and without dose dependency in week 13. Therefore, no toxicological effect of the test item is considered.
Functional observation was not recorded for all dose groups at the end of the recovery period. As the functional observation showed no toxicological effects observed during the treatment period and clinical observation was without toxicological effects during the recovery phase, no toxicological relevant effects are assumed during the recovery of the animals.
For a detailed desription of the findings in tabular form please refer to the attachment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant changes to the control group were found in male animals of the mid- and high-dose groups at the end of the treatment period, but there were no statistically significant differences in organ weight between all female dose groups and the control group with the exception of spleen in the mid-dose group (relative to body weight 0.02% above control).
There were statistically significant differences for brain relative to body weight (mid-dose: 19.83% and high-dose: 24.25% above control), for spleen relative to body weight (high-dose: 0.03% above control), for thymus (absolute high-dose: 21.51% below control, relative to brain weight (mid-dose: 20.80% below control), for testes relative to body weight (mid-dose: 19.10% and high-dose: 24.19% above control) and for kidney (absolute high-dose: 11.13% below control, relative to body weight mid-dose: 8.89% and high-dose: 13.61% above control). Further organ weights with statistical significance in males were seen for pituitary gland (absolute high-dose: 22.35% below control, relative to brain weight 20.17% below control), for adrenal glands relative to body weight (high-dose: 32.72% above control) and heart relative to body weight (high-dose: 10.17% above control). Statistical significance in the male mid-dose and high-dose group was found for the reproductive organs epididymides (absolute 24.65% and 17.37% below control, relative to brain weight 24.97% and 15.12% below control) and prostate (absolute 16.40% and 30.92% below control, relative to brain weight 17.15% and 29.20% below control). Furthermore, the absolute liver weight was statistically significantly decreased (mid-dose: 12.63%, high-dose: 11.44%) and additionally decreased in relation to brain weight (13.07%) in the mid-dose and relatively to body weight increased in the high-dose group (12.98%).
Considering the statistically significant decrease of final body weight in mid-dose and high-dose males, the statistically significant absolute and relative weight changes of several organs in the male groups are considered to be related to the decrease in body weight when compared to control. Furthermore, the histopathological evaluation showed no correlating test item-related findings. Therefore, the statistical significances are considered not to be adverse effects of the test item.
The statistically significant differences relative to body weight at the end of the recovery period in the male and female high-dose groups for brain (male: 15.91% above control), testes (37.72% above control), kidneys (male 17.77%, female: 13.81% above control), adrenal glands (male 11.14%, female 33.65% above control), heart (male 18.22%, female 10.09% above control) and liver (male 11.21% above control) are considered to be an effect of the statistically significant decrease in final body weight. Additionally, the absolute weight of pituitary gland showed a statistically significant decrease in high-dose males (13.78% below control).
For a detailed desription of the findings in tabular form please refer to the attachment.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic findings at the end of the treatment period were red coloured mesenteric lymph nodes in one male and three females of the high-dose group. For one high-dose female, the uterus was dilated and for one low-dose male the thymus was abnormally coloured.
Furthermore, findings were noted for one high-dose female. For this animal the uterus with cervix, oviducts and ovaries were not found at necropsy and the vagina was irregularly shaped, but a testis with normal appearance was found. This finding is considered to be incidental and not related to an effect of the test item.
The necropsy at the end of the recovery period showed no macroscopic findings in all animals of the control and high-dose groups in males and females.
Under consideration of the histopathological evaluation, all observed gross lesions were incidental findings and were considered not related to the treatment with the test item.
For a detailed desription of the findings in tabular form please refer to the attachment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes were observed during the histopathological evaluation.
There was no histological evidence of toxicity in the reproductive organs and tissues including testes, epididymides, prostate gland, seminal vesicles, coagulating glands, ovaries, uterus, cervix and vagina in any of the investigated animals. The testes were checked on completeness of cell populations, completeness of stages and degenerative changes. No treatment-related effects on the testicular histomorphology were observed and the histological appearance reflected the animal physiology. Further, no treatment-related effect on interstitial cell structure was noticed.
Other findings recorded at histopathological evaluation are described in the following.
Mesenteric lymph nodes - minimal to moderate hemorrhages, minimal to slight lymphoid depletion and minimal hemosiderin deposition within macrophages was observed in some control and high-dose animals. The observed hemorrhages and the hemosiderin deposition occurred most likely perimortem and were deemed incidental. The minimal lymphoid depletion was considered to be most likely related to a stressful condition and also deemed to be incidental.
Urinary bladder - minimal hemorrhage was observed in the urinary bladder wall in two high-dose males. This change occurred most likely around the time of death and was deemed to be incidental.
All other findings recorded at histopathological evaluation were deemed to be incidental or were within the range of background alterations that may be observed in animals of this strain and age and in this study type.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

In the daily evaluation of the estrus cycle in the last week of the treatment and recovery period, showed no toxicological relevant differences for all female animals in all dose groups. There were no statistical significances on the testis weight, mean sperm count and mean sperm motility for all dose groups after the treatment period or after the recovery period. Mean total number of abnormal and normal sperms/findings and therefore the relative number of normal and abnormal sperms showed statistical significances after the end of the treatment period in the mid- and high-dose male groups and after the recovery period in high-dose males. For a detailed desription of the findings in tabular form please refer to the attachment.


The relative mean value for normal sperm was slightly decreased in the mid- and high-dose group (mid-dose: 90.95%, high-dose: 91.50%) when compared to control (93.05%) and the relative mean value for abnormal sperm slightly increased (mid-dose: 9.05%, high-dose 8.50%) when compared to control (6.95%). At the end of the recovery period, the relative mean value for normal sperm was 90.50% in the high-dose group and 93.80% in the control group, the relative mean value for abnormal sperm was 9.50% in the high-dose and 6.20% in the control. For a detailed desription of the findings in tabular form please refer to the attachment.
As the histopathological evaluation showed no toxicological findings for male reproductive organs and additionally, the sperm motility showed no abnormal findings, the statistical significances in sperm morphology are considered to be not related to the treatment with test item.

There were no statistically significant changes in hormone analysis (T3, T4, TSH) at the end of the treatment and recovery period. Mean values of all male and female dose groups were comparable to the control group and no toxicological effect of the test item is concluded. For a detailed desription of the findings in tabular form please refer to the attachment.

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

Key result

Critical effects observed:

no

Conclusions:

In a GLP compliant 90-Day Repeated Dose Oral Toxicity study acccording to OECD 408 with hexadecyl(trimethoxy)silane, the no observed adverse effect level (NOAEL) is considered to be 100 mg/kg bw/day based on the reduced male and female body development at 300 mg/kg bw/day (males) and 1000 mg/kg bw/day (males and females) and additional clinical findings in high-dose males and females. At histopathological evaluation, no test item-related changes were observed.

Cross-reference 11

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study planned

Study period:

The test will be conducted after a decision on the requirement to carry out the proposed test has been taken according to the procedure laid down in Regulation (EC) 1907/2006 and a deadline to submit the information required has been set by the Agency.

Justification for type of information:

TESTING PROPOSAL ON VERTEBRATE ANIMALS

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Hexadecyl(trimethoxy)silane (CAS 16415-12-6)

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Available GLP studies: No adequate and reliable GLP studies addressing developmental toxicity in a second species are available with the registration substance.
- Available non-GLP studies: No adequate and reliable non-GLP studies addressing developmental toxicity in a second species are available with the registration substance.
- Historical human/control data: no data available
- (Q)SAR: Currently available Q(SAR) methods are not applicable to assess the full scope of developmental toxicity.
- In vitro methods: No validated in vitro methods to assess developmental toxicity are available so far.
- Weight of evidence: So far there is no information available which is suitable to assess developmental toxicity in a second species in a weight of evidence approach.
- Grouping and read-across: No suitable read-across options are available.

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- In order to fulfil the standard information requirements, a GLP-compliant pre-natal developmental toxicity study in rabbits via the oral route following OECD guideline 414 is proposed according to Annex X, Column 1, Section 8.7.2.
Column 2 adaption possibilities at the Annex X level where considered and do not apply: Based on the available data the registration substance is not regarded as CMR substance. In particular it is not known to be a genotoxic carcinogen or germ cell mutagen. In addition it is not classified for reproductive toxicity Cat 1A or 1B in accordance with Regulation (EC) No. 1272/2008. In addition, there are no toxicokinetic studies available which could justify the absence of systemic absorption.

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
none

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

June 2018

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Route of administration:

oral: gavage

Materials and methods

Test material

Test material information

Constituent 1

Reference substance name:

Hexadecyltrimethoxysilane

EC Number:

240-464-3

EC Name:

Hexadecyltrimethoxysilane

Cas Number:

16415-12-6

Molecular formula:

C19H42O3Si

IUPAC Name:

hexadecyltrimethoxysilane