3-(triethoxysilyl)propyl methacrylate

Endpoint summary

Description of key information

No skin sensitisation data were available for the registered substance, therefore data were read-across from the structural analogous substances, 3-(trimethoxysilyl)methyl methacrylate (CAS 54586-78-6) and 3-(trimethoxysilyl)propyl methacrylate (CAS 2530-85-0).

The key study for skin sensitisation with 3-(trimethoxysilyl)methyl methacrylate found the test material not sensitising in a guideline study which was compliant with GLP (BSL Bioservice, 2003). The study was conducted in accordance with OECD guideline and in compliance with GLP.

In the key mouse local lymph node assay, conducted according to the appropriate OECD 429 Test Guideline and in compliance with GLP, 3-(trimethoxysilyl)propyl methacrylate (CAS 2530-85-0) was reported not to elicit a SI ≥ 3 when tested up to 10% and it was established that the EC3 value exceeds 10%. The test material was concluded to be not sensitising to skin (Charles River, 2018).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

An LLNA study was not performed because there is an existing reliable study for skin sensitisation using the Guinea Pig Maximisation test method. Furthermore, the study pre-dates REACH Regulation (EC No 1907/2006) and CLP Regulation (EC No 1272/2008) requirements, which took place in 2007. According to the requirements the first choice in vivo test method for skin sensitisation is the murine local lymph node assay (LLNA).

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, D33178 Borchen
- Weight at study initiation: 300-500g
- Housing: the animals were kept in groups in Terluran cages on Altromin saw fibre bedding, max group size 10 animals
- Diet: Altromin 3122 maintenance diet for guinea pigs, rich in crude fibre, ad libitum
- Water: tap water, ad libitum
- Acclimation period: described as 'adequate'

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

Route:

intradermal

Vehicle:

corn oil

Concentration / amount:

For the intradermal injection (induction - first stage) the test item was applied at a 2.5% concentration (diluted in corn oil). For the topical application (induction - second stage) the test item was applied at a 100% concentration. For the topical application (challenge) the test item was applied at 100% concentration.

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

corn oil

Concentration / amount:

For the intradermal injection (induction - first stage) the test item was applied at a 2.5% concentration (diluted in corn oil). For the topical application (induction - second stage) the test item was applied at a 100% concentration. For the topical application (challenge) the test item was applied at 100% concentration.

No. of animals per dose:

10/5 (test/control)

Details on study design:

RANGE FINDING TESTS: For the justification of dose levels a preliminary test was performed. Three animals were intradermally treated with 1%, 2.5% and 5% concentration of the test item. For the 1% concentration slight signs of erythema (grade 1) were recorded 24h as well as 48h after application. These symptoms had disappeared 72 h after application. For the 5% concentration erythema grade 1, as well as necrotic formations, was recorded at the 24h, 48h as well as 72 hours reading. For the 2.5% concentration erythema grade 1 was recorded 24h, 48h as well as 72 hours after application.

Therefore, the concentration of 2.5% was chosen for the intradermal induction. Two animals were topically treated with 50% as well as 100% for 24 h and 48 hours respectively. No signs of irritation and systemic toxicity were recorded during the observation time for the animals. Therefore the 100% concentration was chosen for the topical induction as well as for the challenge.

MAIN STUDY
A. INDUCTION EXPOSURE

FIRST STAGE: INTRADERMAL INJECTION

Three pairs of intradermal injections of 0.1ml volume were given in the shoulder region which was cleared of hair by clipping so that one of each pair lies on each side of the midline.

Test group: Day 0
Injection 1: Freund's Adjuvant complete, 1 + 1 (v/v) diluted with isotonic saline
Injection 2: Prepared test item
Injection 3: Prepared test item at a concentration of 50% (v/v) in Freund's Adjuvant complete, 1+1 (v/v) diluted with isotonic saline

Control group: Day 0
Injection 1: Freund's Adjuvant complete, 1+1 (v/v) diluted with isotonic saline
Injection 2: vehicle
Injection 3: vehicle at a concentration of 50% (v/v) in Freund's Adjuvant complete, 1+1 (v/v) diluted with isotonic saline

Injections 1 and 2 were given close to each other and nearest to the head, while 3 is given toward the caudal part of the test area.

SECOND STAGE: TOPICAL APPLICATION

Test and control group: Day 6
Approximately 24 hrs before the topical induction application the test area, after close clipping, was painted with 0.5ml of 10% sodium lauryl sulphate in vaseline, in order to create a local irritation.

Test group: Day 7
A patch was loaded with 0.5ml of the prepared test item, applied to the test area and held in contact by an occlusive dressing for 48 hours.

Control group: Day 7
A patch was loaded with 0.5ml of the vehicle and applied to the test area and held in contact by an occlusive dressing for 48 hours.



B. CHALLENGE EXPOSURE

TOPICAL APPLICATION

The flanks of treated and control animals were cleared of hair by closely clipping and the use of depilation creme.

Test and control group: Day 20

A patch loaded with 0.5ml of the prepared test item was applied to the left flank of the animals and a patch loaded with 0.5ml vehicle to the right flank (intraspecific control), respectively. The patches were held in contact by an occlusive dressing 24 hours.


OBSERVATION

Test and control group

Approximately 21 hours after removing the patch the challenge area was cleaned and cleared of hair. Approximately 24 hours after removing of the patch the skin reaction was observed and recorded according to the grades shown below. Two more observations were recorded 48 and 72 hours after patch removal. Additionally, all animals have been observed for signs of toxicity at least once daily during the test period.

Positive control substance(s):

yes

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100%

No. with + reactions:

0

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:

100%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

100%

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

100%

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

15% Mercaptobenzothiazole

Remarks on result:

other: sensitization rate of 70%

In all animals slight signs of erythema were recorded 24 h as well as 48 hours after first application. No other signs of irritation were observed in any of the animals neither after the intradermal application (induction first stage). No signs of irritation were observed after the topical application (induction second stage).

Challenge readings: The results of the test animals at the challenge phase were compared with the results of the control animals. No signs of irritation were observed after the challenge. The maximum percentage of animals sensitized was 0%. Animals of both groups survived throughout the test period. Animals of the test group showed neither reduced weight gain as compared to historical data and the animals of the control group, nor any other signs of toxicity.

Interpretation of results:

GHS criteria not met

Conclusions:

The test material was found not sensitising in a guideline study which was compliant with GLP.

Executive summary:

Slight signs of erythema were recorded in all animals 24 h and 48 hours after the first application. No other signs of irritation were observed in any other animal neither after the intradermal application (induction first stage), nor after the topical application (induction second stage).

Challenge readings: The results of the test animals at the challenge phase were compared with the results of the control animals. No signs of irritation were observed after the challenge. The maximum percentage of animals sensitized was 0%. Animals of both groups survived throughout the test period. Animals of the test group showed neither reduced weight gain as compared to historical data and the animals of the control group, nor any other signs of toxicity.

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature container flushed with nitrogen
- Stability under test conditions: There was no information available about the stability and solubility of the test item in vehicle.
- Solubility and stability of the test substance in the solvent/vehicle: There was no information available about the stability and solubility of the test item in vehicle.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily and dosed within 4 hours after adding the vehicle to the test item. The dosing formulations were kept at room temperature until dosing. The dosing formulations were stirred until and during dosing.
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: not specified
- Age at study initiation: Young adult animals (approximately 10 weeks old)
- Weight at study initiation: 20.4 to 24.1 g.
- Housing: Animals were group housed in polycarbonate cages.
- Diet: Pelleted rodent diet ad libitum
- Water: Municipal tap-water ad libitum
- Acclimation period: at least 5 days
- Indication of any skin lesions: none reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 to 22°C
- Humidity (%): 42 to 46%
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

2, 5 or 10% w/w
The vehicle was chosen from the vehicles specified in the test guideline (in order of preference): Acetone/Olive oil (4:1 v/v), N,N-dimethylformamide, methylethylketone, propylene glycol, dimethylsulfoxide and 1% Pluronic© L92 in Elix water (in case an aqueous vehicle is suitable). The vehicle was selected on the basis of maximizing the solubility based on trial preparations performed at Charles River Den Bosch and on information provided by the Sponsor.

No. of animals per dose:

5 females

Details on study design:

PRE-SCREEN TESTS: A pre-screen test was conducted in order to select the highest test item concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation as the most pronounced response (maximum grade 2 and/or an increase in ear thickness < 25%) and/or is the highest possible concentration that can technically be applied.
- Compound solubility: yes
- Irritation: yes
- Systemic toxicity: yes
- Ear thickness measurements: Ear thickness measurements were conducted using a digital thickness gauge prior to dosing on Days 1 and 3, and on Day 6.
- Erythema scores: not specified.

MAIN STUDY: Three groups of five animals were treated with one test item concentration per group. The highest test item concentration was selected from the pre-screen test. One group of five animals was treated with the vehicle. Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Postdose observations were performed once daily on Days 1-6. Animals were weighed individually on Day 1 (predose) and 6 (prior to necropsy). Erythema and eschar formation observations were performed once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing).

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: local lymph node assay
- Criteria used to consider a positive response: A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean. If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer.


TREATMENT PREPARATION AND ADMINISTRATION:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily and dosed within 4 hours after adding the vehicle to the test item. The dosing formulations were kept at room temperature until dosing. The dosing formulations were stirred until and during dosing. During induction, the dorsal surface of both ears was topically treated (25 μL/ear) with the test item, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing. The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item. Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) containing 20 μCi of 3H-methyl thymidine. After five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) of Euthasol® 20%. The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded.

Positive control substance(s):

other: not included for animal welfare reasons

Statistics:

not used

Positive control results:

No positive control was used in the study.

Key result

Parameter:

SI

Value:

0.9

Test group / Remarks:

2% concentration

Key result

Parameter:

SI

Value:

1.2

Test group / Remarks:

5% concentration

Key result

Parameter:

SI

Value:

0.9

Test group / Remarks:

10% concentration

Key result

Parameter:

EC3

Value:

> 10

Cellular proliferation data / Observations:

PRE-SCREEN TEST: At a 100%, 50% and 25% test item concentration, variation in ear thickness during the observation period were more than 25% from Day 1 pre-dose values and/or clinical signs of systemic toxicity were noted. Therefore, these concentrations did not meet the selection criteria. At a 10% test item concentration no signs of toxicity or excessive irritation were noted and therefore 10% was selected as the highest concentration to be used in the main study.

CELLULAR PROLIFERATION DATA: The majority of the auricular lymph nodes were considered normal in size, except for one node of one animal treated at a 2% test item concentration, which was considered to be enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals.


DETAILS ON STIMULATION INDEX CALCULATION: Mean DPM/animal values for the experimental groups treated with test item concentrations 2, 5 and 10% were 517, 656 and 488 DPM, respectively. The mean DPM/animal value for the vehicle control group was 550. The SI values calculated for the test item concentrations 2, 5 and 10% were 0.9, 1.2 and 0.9, respectively.

EC3 CALCULATION: Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 10%, 3-(Trimethoxysilyl)propyl methacrylate (CAS 2530-85-0) was not considered to be a skin sensitizer. It was established that the EC3 value exceeds 10%.

CLINICAL OBSERVATIONS: No irritation was observed in any of the animals. No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study.

BODY WEIGHTS: Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Interpretation of results:

GHS criteria not met

Conclusions:

In the mouse local lymph node assay, conducted according to the appropriate OECD 429 Test Guideline and in compliance with GLP, 3-(trimethoxysilyl)propyl methacrylate (CAS 2530-85-0) was reported not to elicit a SI ≥ 3 when tested up to 10% and it was established that the EC3 value exceeds 10%. The test material was concluded to be not sensitising to skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

No skin sensitisation data were available for the registered substance, therefore data were read-across from the structural analogous substance, 3-(trimethoxysilyl)methyl methacrylate (CAS 54586-78-6) and 3-(trimethoxysilyl)propyl methacrylate (CAS 2530-85-0).

The key study for skin sensitisation found the test material, 3-(trimethoxysilyl)methyl methacrylate, not sensitising in a guideline study which was compliant with GLP (BSL Bioservice, 2003).

Slight signs of erythema were recorded 24 hours as well as 48 hours in all animals after first application. No other signs of irritation were observed in any animal after the intradermal application (induction first stage). No signs of irritation were observed after the topical application (induction second stage).

The results of the test animals at the challenge phase were compared with the results of the control animals. No signs of irritation were observed after the challenge. The maximum percentage of animals sensitized was 0%. Animals of both groups survived throughout the test period. Animals of the test group showed no reduced weight gain as compared to historical data and the animals of the negative control group, or any other signs of toxicity.

The sensitising potential of 3-(trimethoxysilyl)propyl methacrylate (CAS 2530-85-0) was assessed in a mouse local lymph node assay, conducted according to the appropriate OECD 429 Test Guideline and in compliance with GLP (Charles River, 2018). Three experimental groups of five female mice were treated with test item concentrations of 2, 5 or 10% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Acetone/Olive oil (4:1 v/v)). The vehicle was chosen from the vehicles specified in the test guideline (in order of preference): acetone/olive oil (4:1 v/v), N,N-dimethylformamide, methylethylketone, propylene glycol, dimethylsulfoxide and 1% Pluronic© L92 in Elix water (in case an aqueous vehicle is suitable). The vehicle was selected on the basis of maximizing the solubility based on trial preparations performed at Charles River Den Bosch and on information provided by the Sponsor. Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal.  After precipitating the DNA of the lymph node cells, radioactivity measurements were performed.

No irritation was observed in any of the animals. The mean body weight gain shown by the animals over the study period was considered to be normal. The majority of the auricular lymph nodes were considered normal in size, except for one node of one animal treated at a 2% test item concentration.  No macroscopic abnormalities of the surrounding area were noted for any of the animals.

3-(Trimethoxysilyl)propyl methacrylate (CAS 2530-85-0) was reported not to elicits a SI ≥ 3 when tested up to 10% and it was established that the EC3 value exceeds 10%. The test material was concluded to be not sensitising to skin.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available read-across data, no classification is required for skin sensitisation in accordance with Regulation (EC) No. 1272/2008.