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

Endpoint summary

Description of key information

The potential for skin sensitisation was tested in vivo according to OECD 406. None of the test animals showed positive signs of sensitisation, indicating that heptamethylethyltrisiloxane is not a skin sensitizer under the conditions of this study. This conclusion is supported by the results of a DPRA, conducted according to OECD guideline 442C, which showed that heptamethylethyltrisiloxane does not interact with protein moieties.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

23-April-2021 to 30-April-2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation Assays addressing the Adverse Outcome Pathway key event on covalent binding to proteins)

Version / remarks:

26 June 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

Storage conditions: At ambient temperature (15 to 25°C), protected from light

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

Assessment of Test Item Solubility: The solubility of 3-Ethylheptamethyltrisiloxane was assessed at a concentration of 100 mM in acetonitrile.

Preparation of Peptide Stock Solutions: Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in ca 20 mL aliquots of the appropriate buffer solution (Cysteine in 100 mM phosphate buffer pH 7.5, Lysine in 100 mM Ammonium acetate buffer pH 10.2).

Preparation of Peptide Calibration Standards: Calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate buffer and acetonitrile and contained each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 mM. A buffer blank was also prepared.

Preparation of Stability Controls and Precision Controls: Precision controls (Reference Control A) and stability controls (Reference Control B) of both peptides were prepared at a peptide concentration of 0.5 mM. Reference Control A and Reference Control B were buffer and acetonitrile, respectively.

Preparation of Positive Control Solution and Test Item Stock Solution: The positive control chemical (Cinnamic Aldehyde) was prepared at a concentration of 100 mM in acetonitrile. A 100 mM stock solution of 3-Ethylheptamethyltrisiloxane was prepared in acetonitrile.

Preparation of Positive Control and Cysteine Peptide Depletion Samples and Co-elution Controls: Accurate volume aliquots of 3-Ethylheptamethyltrisiloxane and the positive control were diluted with the Cysteine peptide stock solution to prepare solutions containing 0.5 mM Cysteine and 5 mM of either 3-Ethylheptamethyltrisiloxane or the positive control. For the co-elution control, buffer solution was used in place of the Cysteine stock solution.

Preparation of Positive Control and Lysine Peptide Depletion Samples and Co-elution Controls: Accurate volume aliquots of 3-Ethylheptamethyltrisiloxane and the positive control were diluted with the Lysine peptide stock solution to prepare solutions containing 0.5 mM Lysine and 25 mM of either 3-Ethylheptamethyltrisiloxane or the positive control. For the co-elution control, buffer solution was used in place of the Lysine stock solution.

Incubation: The appearance of the 3-Ethylheptamethyltrisiloxane and positive control samples in the HPLC vials was documented after preparation and then the vials were placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to injection of the samples as part of an analytical run. Before initiation of the run the appearance of the samples in the vials was assessed and documented again.

Analysis: The concentration of both the Cysteine and Lysine peptides in the presence of 3-Ethylheptamethyltrisiloxane and the associated positive controls was quantified by HPLC using UV detection as detailed in the chromatographic section.

Calculations: The peak area response for the peptide in each calibration chromatogram was measured. Calibration curves were constructed by linear regression of standard response versus standard concentration. The area responses of the peptide peak observed at the characteristic retention time of each peptide in each sample chromatogram was measured. Peptide depletion was determined using the following equation: % Peptide depletion = 100 - Peptide peak area in replicate depletion samples (x 100)/Mean Peptide peak area of reference control samples B or C.

Vehicle / solvent:

acetonitrile

Positive control:

cinnamic aldehyde

Positive control results:

The positive control, cinnamic aldehyde, achieved a mean of 71.2% ±1.04
and 56.8% ±1.11 in the Cysteine and in the Lysine depletion assay, respectively.

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

cysteine depletion

Value:

5.1 %

At concentration:

0.5 mM

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

lysine depletion

Value:

-0.9 %

At concentration:

0.5 mM

Vehicle controls validity:

valid

Positive controls validity:

valid

Outcome of the prediction model:

negative [in vitro/in chemico]

Other effects / acceptance of results:

The solubility of 3-Ethylheptamethyltrisiloxane in acetonitrile at a nominal concentration of 100 mM was achieved. There were no co-elution peaks in either the Cysteine or Lysine assays.

Applying the depletion model, with the mean of Cysteine and Lysine depletion at 2.1%, the reactivity of 3-Ethylheptamethyltrisiloxane is classed as “no or minimal reactivity”.
The DPRA prediction is, therefore, negative and 3-Ethylheptamethyltrisiloxane is not predicted to be a sensitizer by this assay.

Table 1: Overall Achieved Depletion Values

 

Test item	Cysteine peptide depletion (%)	Lysine peptide depletion (%)	Overall mean depletion (%)	Reactivity class	DPRA prediction
3-Ethylheptamethyltrisiloxane	5.1	-0.9	2.1	No or minimal	Negative

 

Table 2 - Individual Achieved Depletion Values

Table 2A: Cysteine Peptide Depletion

Sample	Peak area (µV.sec)	Peptide concentration^1 (µg/mL)	Peptide Depletion^2 (%)	Mean Depletion (%)	SD (%)
Positive control	282279	110	70.0	71.2	1.04
	291159	103	71.7
	297169	103	71.8
3-Ethylheptamethyltrisiloxane	608950	359	3.2	5.1	2.87
	576477	340	8.4
	606500	358	3.5

SD       Standard Deviation

¹          Samples prepared at a nominal concentration of 376 µg/mL (0.5 mM)

²          Calculated against a mean Reference Control B area of 629280 µV.sec (n=6)

 

Table 2B: Lysine Peptide Depletion

 

Sample	Peak area (µV.sec)	Peptide concentration^1 (µg/mL)	Peptide Depletion^2 (%)	Mean Depletion (%)	SD (%)
Positive control	282279	164	58.0	56.8	1.11
	291159	169	56.7
	297169	172	55.8
3-Ethylheptamethyltrisiloxane	678113	389	-0.9	-0.9	1.13
	685429	394	-2.0
	670210	385	0.3

SD       Standard Deviation

¹          Samples prepared at a nominal concentration of 388 µg/mL (0.5 mM)

²          Calculated against a mean Reference Control B area of 671890 µV.sec (n=6)

Interpretation of results:

study cannot be used for classification

Conclusions:

In a DPRA conducted according to OECD guideline 442C and under GLP principles, 3-Ethylheptamethyltrisiloxane showed minimal reactivity with cysteine and lysine moieties (mean of Cysteine and Lysine % depletion at 2.1%). Based on the prediction model 3-Ethylheptamethyltrisiloxane is placed in the reactivity class of “no or minimal” and hence it is predicted to be negative in terms being a potential skin sensitizer.

Executive summary:

The reactivity and sensitizing potential of 3-Ethylheptamethyltrisiloxane was assessed using the Direct Peptide Reactivity Assay (DPRA), according to OECD/OCDE document TG 442C and following GLP principles.

 

Solutions of 3-Ethylheptamethyltrisiloxane were analyzed by the DPRA analytical method in both Cysteine and Lysine containing synthetic peptides. 3-Ethylheptamethyltrisiloxane achieved a concentration of 5mM and 25mM in the cysteine and lysine stock solution, respectively. Acetonitrile was used as solvent.

 

The results of the control substances demonstrated validity of the assays. The peptide depletion by the test item reached a mean of 5.1% in the cysteine assay and -0.9% in the lysine assay. Toghether, the mean of Cysteine and Lysine depletion was 2.1%. 

 

Applying a depletion model, with the mean of Cysteine and Lysine depletion at 2.1%, the reactivity of 3-Ethylheptamethyltrisiloxane is classed as “no or minimal reactivity”. Under the conditions of this study, the DPRA prediction is negative and 3-Ethylheptamethyltrisiloxane is not predicted to be a sensitizer under the conditions of this assay.

Reference 2

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4 March 2003 to 28 March 2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

September 30, 1996

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

July 17, 1992

Deviations:

yes

Remarks:

the dose used to challenge was non-irritant

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The test was performed before LLNA was implemented.

Specific details on test material used for the study:

Density: 828 g/L at 20°C

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

Test animals:
Age:5 weeks at study start
Weight: not reported
Acclimatisation:13 days
Housing: group housed (maximum 10 animals/cage)
Diet: standard laboratory diet ad libitum
Water: tap water ad libitum

Environmental conditions:
Temperature: 20 +/-3°C
Humidity: 30-70%
Ventilation: 10 air changes/hour
Lighting: 12 hours light/dark cycle

Route:

intradermal

Vehicle:

maize oil

Concentration / amount:

10%

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Route:

intradermal

Vehicle:

other: FCA/test substance in vehicle 1:1

Concentration / amount:

10%

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Day(s)/duration:

2

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

maize oil

Concentration / amount:

30%

Day(s)/duration:

1

Adequacy of challenge:

other: non-irritant concentration

No. of animals per dose:

Test group: 10 animals
Control group: 5 animals

Details on study design:

The dose levels were chosen following a preliminary irritation study using two animals per treatment route:
Intradermal injections at concentrations of 1,3,10, 30 and 100% and topical treatment at 30 and 100%.
Intradermal injection at concentrations of 30 and 100% produced abscesses
Topical treatment at 100% produced moderate and confluent erythema

Challenge controls:

Control groups were treated with FCA/physiological saline or vehicle only as appropriate during the induction phase and vehicle only during the challenge phase

Positive control substance(s):

yes

Remarks:

positive control study run concurrently using 20 and 10% dilution of hexylcinnamaldehyde in saline induced positive reactions in all animals.

Positive control results:

The study with positive control alpha-hexylcinnamaldehyde (application of a 10% dilution) was run in March 2003 in the same test facility. All 5 test animals showed positive response at 24 and 48 hours, while the three animals in the negative control group did not show a reaction.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

negative control

No. with + reactions:

2

Total no. in group:

5

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

negative control

No. with + reactions:

0

Total no. in group:

5

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

30%

No. with + reactions:

2

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

30%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Reading:

other:

Group:

positive control

Remarks on result:

positive indication of skin sensitisation

Remarks:

Positive control not run simultaneously

Maximum concentration not causing irritating effects in preliminary test: 30 %

Signs of irritation during induction:
Moderate erythema

Evidence of sensitisation of each challenge concentration:
Control group: 2 of 5 animals showed slight erythema after 24 hours; after 48 hours no effects were observed.

Test group: 2 of 10 animals showed slight erythema after 24 hours; after 48 hours no effects were observed.

Other observations:
None

Interpretation of results:

GHS criteria not met

Conclusions:

In a guinea pig maximization study, performed according to OECD/EC guidelines and under GLP principles, none of the test animals showed positive signs of sensitisation. A positive control group demonstrated the sensitivity of the test system. Based on these findings, it is concluded that heptamethylethyltrisiloxane is not a skin sensitizer under the conditions of this study.

Executive summary:

The skin sensitization potential of heptamethylethyltrisiloxane was evaluated in female guinea pigs using the Maximization Test. Test substance concentrations used in the main study were based on an initial irritancy screen.

The first induction was performed by intradermal injection with 10% test substance suspension in maize oil and an emulsion of Freund's complete adjuvant (FCA)/10% test substance in maize oil. One week later, a second induction was made by topical application (48 hours) under an occlusive dressing of undiluted test material. Two weeks after the second induction, the animals were challenged by topical application of 30% test substance ointment in maize oil under an occlusive dressing for 24 hours.

Both test and control animals reacted slightly to the test substance and to the vehicle alone at 24 hours after challenge. The degree and incidence were comparable for both test and control groups and considered to be signs of primary skin irritation. None of the test animals showed positive signs of sensitisation. A positive control group demonstrated the sensitivity of the test system.

Based on these findings, it is concluded that heptamethylethyltrisiloxane is not a skin sensitizer under the conditions of this study

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on all data available, no classification is warranted for skin sensitisation according to CLP (1272/2008/EC) classification criteria.