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Diss Factsheets

Administrative data

Description of key information

overall: not sensitising

weight of evidence:

- positive in GPMT study (positive reactions in 7/20 animals)

- inconclusive in LLNA, but clearly negative in follow up study with challenge

- supporting data on structurally related substances: negative

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar physicochemical, ecotoxicological and toxicological properties because
• they are manufactured from similar or identical precursors under similar conditions
• they share structural similarities with common functional groups: methacrylate esters
• the metabolism pathway leads to comparable products (methacrylic acid and medium chain alcohol).

Therefore, read-across from the existing physicochemical, ecotoxicity and toxicity studies on the source substances is considered as an appropriate adaptation to the standard information requirements of REACH regulation

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see “Justification for read-across” attached to IUCLID section 13

3. ANALOGUE APPROACH JUSTIFICATION
see “Justification for read-across” attached to IUCLID section 13

4. DATA MATRIX
see “Justification for read-across” attached to IUCLID section 13
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across: supporting information
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Key result
Parameter:
EC3
Remarks on result:
other: The EC3 value could not be determined, since all S.I. value were <3.
Key result
Parameter:
SI
Value:
< 3
Test group / Remarks:
highest tested concentration 25%
Remarks on result:
other: not sensitising
Parameter:
SI
Value:
0.9
Test group / Remarks:
2.5%
Parameter:
SI
Value:
>= 0.9 - <= 0.99
Test group / Remarks:
5%
Parameter:
SI
Value:
>= 1.9 - <= 2.11
Test group / Remarks:
10%
Parameter:
SI
Value:
2.66
Test group / Remarks:
25%
Interpretation of results:
GHS criteria not met
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
adopted 17 July 1992
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The study was conducted before the new annex of the REACH Regulation entered into force and is used in a weight-of-evidence approach.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
not specified
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: David Hall Limited, Burton-on-Trent, Staffordshire, UK
- Age at study initiation: approx. eight to twelve weeks
- Weight at study initiation: 308 to 404g
- Housing: singly or in pairs in solid-floor polypropylene cages furnished with woodflakes
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 21°C
- Humidity (%): 47 to 57 %
- Air changes (per hr): approx. 15
- Photoperiod (hrs dark / hrs light): 12/12
Route:
intradermal and epicutaneous
Vehicle:
arachis oil
Concentration / amount:
Intradermal Induction: 25% w/v in arachis oil BP
Topical Induction: undiluted as supplied
Day(s)/duration:
Intradermal: day 1; topical: day 7, 48 h
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:
day 21
Adequacy of challenge:
highest non-irritant concentration
Route:
epicutaneous, occlusive
Vehicle:
arachis oil
Concentration / amount:
75%
Day(s)/duration:
day 21
No. of animals per dose:
main study: 20 (test group) + 10 (control group)
Details on study design:
RANGE FINDING TESTS:
Selection of Concentration tor Intradermal Induction:
A total of four guinea pigs were used, each guinea pig receiving four 0.1 ml injections of one concentration of test material (25%, 10%, 5% and 1% w/v in arachis oil BP). The degree of erythema at the injection sites was assessed approximately 24, 48 and 72 hours and 7 days after injection according to the Draize scale. The degree of oedema was not evaluated. Any evidence of systemic toxicity was also recorded. The highest concentration that caused only mild to moderate skin irritation, and wh ich was weil tolerated systemicall y, was selected for the intradermal induction stage of the main study.

Selection of Concentration tor Topical Induction
Two guinea pigs (intradermally injected with Freund's Complete Adjuvant 15 days earlier) were treated with the undiluted test material and three preparations of the test material (75%, 50% and 25% v/v in arachis oil BP). Applications were made to the clipped flanks under occlusive dressings for an exposure period of 48 hours. The degree of erythema and oedema was evaluated approximately 1, 24 and 48 hours after dressing removal. The highest concentration producing only mild to moderate dermal irritation was selected for the topical induction stage of the main study.

Selection of Concentration for Topical Challenge
The undiluted test material and three preparations of the test material (75%, 50% and 25% v/v in arachis oil BP) were applied to the clipped flanks of two guinea pigs under occlusive dressings for an exposure period of 24 hours. These guinea pigs did not form part of the main study but had been treated identically to the control animals of the main study, up to Day 14. The degree of erythema and oedema was evaluated approximately 1, 24 and 48 hours after dressing removal. The highest non-irritant concentration of the test material and one lower concentration were selected for the topical challenge stage of the main study. Very slight (grade 1) erythma was noted in both animals after 1 h at 75 and 100% test item concentration, which had resolved at the 24 h observation.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 1
- Test group
Freund's Complete Adjuvant plus distilled water in the ratio 1:1
a 25% w/v formulation of the test material in arachis oil BP
a 25% w/v formulation of the test material in a 1:1 preparation of Freund's Complete Adjuvant plus distilled water.
One week later (Day 7), the same area on the shoulder region used previously for intradermal injections was clipped again and treated with a topical application of the undiluted test material (48 h, occlusive).
- Control group:
Freund's Complete Adjuvant plus distilled water in the ratio 1:1
arachis oil BP
a 50% w/v formulation of arachis oil BP in Freund's Complete Adjuvant/distilled water 1:1
The topical applications followed the same procedure as for the test animals except that nothing was applied to the filter paper (day 7, 48 h, occlusive).

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: day21
- Exposure period: 24
- Test groups: undiluted (right flank), 75% v/v in arachis oil BP (left flank)
- Control group: undiluted (right flank), 75% v/v in arachis oil BP (left flank)
- Evaluation (hr after challenge): 24 and 48 hours after challenge dressing removal
Challenge controls:
yes
Positive control substance(s):
yes
Remarks:
2-mercaptobenzothiazole
Positive control results:
2-mercaptobenzothiazole in arachis oil (tested in May/June 1989) produced a sensitisation rate of 100%
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
100%
No. with + reactions:
7
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
100%
No. with + reactions:
2
Total no. in group:
20
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
100%
No. with + reactions:
2
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
75%
No. with + reactions:
0
Total no. in group:
20
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
50 and 25% 2-mercaptobenzothiazole
No. with + reactions:
10
Total no. in group:
10
Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The test material produced a 35% (7/20) sensitisation rate and was classified as a moderate sensitiser to guinea pig skin.
Executive summary:

In a dermal sensitisation study according to OECD TG 406, 1992) with Isotridecyl methacrylate, young adult Dunkin-Hartley guinea pigs were tested using the Maximization test method.

Intradermal Induction was conducted with 25% w/v test item in arachis oil BP. For topical induction, the test item was used undiluted as supplied. Two challenge concentrations were used: undiluted as supplied and 75% v/v in arachis oil BP.

Very slight to well-defined erythema was noted at the intradermal induction sites of all test group animals at the 24 and 48-hour observations. Very slight erythema was noted at the intradermal induction sites of all control group animals at the 24-hour observation and in six control group animals at the 48-hour observation.

After topical induction, very slight to well-defined erythema, with or without very slight to slight oedema, were noted at the induction sites of all test group animals at the 1 and 24-hour observations. Bleeding from the intradermal induction sites was noted in fifteen test group animals at the 1-hour observation. Small superficial scattered scabs were noted at the induction sites of three test group animals at the 24-hour observation.

Very slight to well-defined erythema and an isolated incident of very slight oedema were noted at the treatment sites of all control group animals at the l-hour observation. Bleeding from the intradermal induction sites was noted at the treatment site of one control group animal. No skin reactions were noted at the treatment sites of control group animals at the 24-hour

observation.

 

After challenge with undiluted test item positive skin responses (very slight to well-defined erythema - grades 1 to 2) and incidents of very slight oedema were noted at the challenge sites of

seven test group animals at the 24-hour observation and in two test group animals at the 48-hour observation.

Challenge with 75% test item in Arachis Oil lead to positive skin responses (very slight to well-defined erythema - grades 1 to 2) and incidents of very slight oedema were noted at the challenge sites of four test group animals at the 24-hour observation and persisted in one test group animal at the 48-hour observation.

No skin reactions were noted at the challenge sites of control group animals at the 24 or 48-hour observations with either 100% or 75% test item concentration.

 

The positive control substance was 2-mercaptobenzothiazole with a sensitisation rate of 100 %.

 

The test item Isotridecyl methacrylate produced a 35% (7/20) sensitisation rate and was classified as a moderate sensitiser to guinea pig skin. 

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

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:
24 April 2002
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 18.0 - 24.5 g
- Housing: individually
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 days
Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 45-65%
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25, 50, and 100% in acetone:olive oil (4+1)
No. of animals per dose:
5
Details on study design:
MAIN STUDY
TREATMENT PREPARATION AND ADMINISTRATION:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear (left and right) with different test item concentrations of 25, 50, and 100% (w/v) in acetone:olive oil (4+1). The application volume, 25 μl, was spread over the entire dorsal surface (approx. 8 mm) of each ear once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).
Five days after the first topical application, all mice were administered with 250 μl of 80.9 μCi/ml 3HTdR (corresponds to 20.22 μCi 3HTdR per mouse) by intravenous injection via a tail vein.
Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital-Natrium.
The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per animal). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze.

A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
- First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index.
- Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.
Parameter:
SI
Value:
5.86
Test group / Remarks:
25%
Parameter:
SI
Value:
5.52
Test group / Remarks:
50%
Parameter:
SI
Value:
6.83
Test group / Remarks:
100%
Cellular proliferation data / Observations:
EC3 CALCULATION
The EC3 value could not be calculated, since all S.I.s are above 3.

CLINICAL OBSERVATIONS:
No deaths occurred during the study period. No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

BODY WEIGHTS
The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

 

Animal number

DPM

values

measured

DPM−BG per animal

(2 lymph nodes) a)

Mean DPM

S.I. b)

Mean S.I.

Background

1

13

---

---

---

---

 

2

12

---

---

---

---

control

1

705

693

381.9

---

1.00

 

2

640

628

---

 

3

1178

1166

---

 

4

731

719

---

 

5

627

615

---

25%

1

2975

2963

2237.7

3.9

5.86

 

2

3499

3487

4.6

 

3

4804

4792

6.3

 

4

6167

6155

8.1

 

5

4994

4982

6.5

50%

1

4654

4642

2106.3

6.1

5.52

 

2

4402

4390

5.7

 

3

4015

4003

5.2

 

4

3795

3783

5.0

 

5

4259

4247

5.6

100%

1

3623

3611

2607.7

4.7

6.83

 

2

3891

3879

5.1

 

3

5966

5954

7.8

 

4

6602

6590

8.6

 

5

6057

6045

7.9

 

a) = values corrected for mean background value.

b) = Stimulation Indices relative to the mean of the control group

Interpretation of results:
other: inconclusive
Conclusions:
On the basis of the S.I. values, the test item Isotridecyl methacrylate would have to be classified as a skin sensitiser. But due to the unclear dose response relationship and the potentially irritating effects, a definite conclusion cannot be reached. Thus, the outcome of this study is considered to be inconclusive. Further evidence regarding the question whether the obtained effect is truly of a sensitising nature can be obtained using the LLNA with Challenge.
Executive summary:

In this study the test item Isotridecyl methacrylate dissolved in acetone:olive oil (4+1) was assessed for its possible contact allergenic potential using the Local Lymph Node Assay (LLNA) in mice in accordance with OECD Guideline 429 (2002). The principle of the LLNA is that sensitisers induce a primary proliferation of lymphocytes in the lymph node draining the application site. The ratio of proliferation in test item treated groups compared to that in vehicle controls is referred to as the Stimulation Index (S.I.). Radioactive labelling is used to measure cell proliferation. A test item is regarded as a sensitiser in the LLNA if exposure to one or more test item concentration results in a 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated test item concentration required to produce a S.I. of 3 is referred to as the EC3 value.

The main experiment was performed using test item concentrations of 25, 50, and 100%.

The animals did not show any signs of local irritation or systemic toxicity during the course of the study and no cases of mortality were observed.

In this study Stimulation Indices (S.I.) of 5.86, 5.52, and 6.83 were determined with the test item at concentrations of 25, 50, and 100% in acetone:olive oil (4+1), respectively. At all tested concentrations a statistically significant difference of DPM per animal was determined in comparison to the vehicle control group (p <0.001). Even though a clear dose-response was not obtained, all S.I. values were well above the threshold of 3.

However, a statistically significant and dose dependent increase in ear weights was obtained in all test item treated groups in comparison to the vehicle control group (p<0.001). Therefore, the potential skin irritating properties of the test item may have contributed to the observed increase in lymph node cell proliferation.

On the basis of the S.I. values, the test item Isotridecyl methacrylate would have to be classified as a skin sensitiser. But due to the unclear dose response relationship and the potentially irritating effects, a definite conclusion cannot be reached. Thus, the outcome of this study is considered to be inconclusive. Further evidence regarding the question whether the obtained effect is truly of a sensitising nature can be obtained using the LLNA with challenge.

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

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:
24 April 2002
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 18.0 - 24.5 g
- Housing: individually
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 days
Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 40-90%
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
12.5 and 25%; sensitization phase
25%; challenge phase
No. of animals per dose:
5
Details on study design:
MAIN STUDY
TREATMENT PREPARATION AND ADMINISTRATION:
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear (left and right) with test item at different concentrations. The application volume, 25 μl, was spread over the entire dorsal surface (approx. 8 mm) of each ear once daily for three consecutive days. Three further groups of mice (vehicle control groups for the sensitization phase (group 1) and the challenge phase (group 2) and the challenge control (group 3)) were treated with an equivalent volume of the vehicle alone. However, animals belonging to the challenge control (group 3) received the test item at a concentration of 25% during the challenge phase. All animals were treated as described on day 1 to 3. Animals of groups 2, 3, and 6 were treated additionally on day 16.
Prior to the first application of the test item and prior to the challenge application on day 16 as well as prior to the application of 3HTdR (days 6 or 16), the ear thickness of all animals was determined using a micrometer.
On day six all mice of groups 1, 4 and 5 were administered with 20.5 μCi of 3HTdR by intravenous injection via a tail vein with 250 μl of 82.0 μCi/ml 3HTdR. On day eighteen all mice of groups 2, 3, and 6 were administered with 20.4 μCi of 3HTdR by intravenous injection via a tail vein with 250 μl of 81.7 μCi/ml 3HTdR.
Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital-Natrium.
The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per animal). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze.

A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
- First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index.
- Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Parameter:
SI
Value:
3.06
Test group / Remarks:
sensitisation phase: 25 % test item concentration, 5 animals
Parameter:
SI
Value:
1.43
Test group / Remarks:
challenge phase: 25% test item concentration, 5 animals
Remarks on result:
other: since this was a follow up study, only one concentration was tested
Parameter:
SI
Value:
1.42
Test group / Remarks:
Challenge Control Group; Challenge Phase (25% test item concentration), 5 animals
Cellular proliferation data / Observations:
EC3 CALCULATION
The EC3 value after challenge could not be calculated, since the S.I. was below 3.

CLINICAL OBSERVATIONS:
No deaths occurred during the study period. The animals did not show signs of systemic toxicity during the course of the study and no cases of mortality were observed.

BODY WEIGHTS
The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Ear Thickness and Ear Weight
In the sensitization phase, a statistically significant increase was observed in the ear weights of the group treated with 25% test item concentration (day 1-3) in comparison to the vehicle control group and also to the group treated with 12.5% test item concentration (day 1-3) (p<0.001). In the challenge phase, a significant increase in ear weights was not observed in the test item treated group in comparison to the control group. However, this was not reflected in the ear thickness measurements where no significant difference was seen between the ear thickness measured in test item treated animals and control animals.

 

Group

Treatment

day 1-3 with

Treatment

day 16 with

Day of

Preparation

Number

of

LN

DPM / LN

*

S.I.**

Sensitisation phase

1 (Vehicle

Control Group;

Sensit. Phase)

Acetone:olive

oil (4+1)

---

6

10

247.6

1.00

 

4 (Test Group;

Sensit. Phase)

12.5% Test

Item

Concentration

---

6

10

334.5

1.35

 

5 (Test Group;

Sensit. Phase)

25% Test

Item

Concentration

---

6

10

756.9 S

3.06

Challenge phase

2 (Vehicle

Control Group;

Challenge

Phase)

Acetone:olive

oil (4+1)

Acetone:olive

oil (4+1)

18

10

174.3

1.00

 

3 (Challenge

Control Group;

Challenge

Phase)

Acetone:olive

oil (4+1)

25% Test

Item

Concentration

18

10

248.0

1.42

 

6 (Test Group;

Challenge

Phase)

25% Test

Item

Concentration

25% Test

Item

Concentration

18

10

249.3

1.43

--- no treatment performed

LN= lymph node

* DPM/node was determined by dividing the sum of the measured values from all lymph nodes

within a group by the number of lymph nodes taken from that group

** S.I. values calculated by dividing the mean DPM of a test group by the mean DPM obtained for

the respective control group

S Statistically significant increase in comparison to the control (vehicle) group (p=0.001)

Interpretation of results:
GHS criteria not met
Conclusions:
The test item Isotridecyl methacrylate is considered not a skin sensitiser under the test conditions of this study.
Executive summary:

The purpose of this Local Lymph Node Assay was to differentiate whether a lymphocyte proliferation observed in a previous study (Harlan CCR Study 1341400) after treatment with Isotridecyl methacrylate was due to a true sensitizing effect or only due to irritation.

The principle of the assay is based on the immunological memory function of a sensitizing agent, which does not occur in irritation reactions.

Based on the results of the above mentioned study, 25% was chosen for evaluation in the Challenge-LLNA because at this concentration a lymphocyte proliferation above the threshold level of three was obtained without eliciting excessive irritation. 12.5% was included as an additional concentration for evaluation in the sensitization phase because it was expected that at this concentration, the test item would not have an irritative potential.

The present study was divided into 2 treatment phases, the sensitization phase and the challenge phase. Altogether, 6 groups each of five female mice were treated with the vehicle or the test item at different concentrations by topical application at the dorsum of each ear once daily each on three consecutive days:

A vehicle control group (acetone:olive oil (4+1)) for the sensitization phase, two test item groups for the sensitization phase treated with 12.5% and 25% test item concentration, a vehicle control group for the challenge phase which was treated with the vehicle only both in the sensitization phase and the challenge phase, a challenge control group which received vehicle only in the sensitization phase of the experiment and 25% test item in the challenge phase, and a test item group for the challenge phase treated with 25% test item concentration both in the sensitization phase and the challenge phase.

In the first phase (sensitization phase) of the study, on day 6 of the experiment the groups treated with test item concentrations of 12.5% and of 25% and one vehicle group were used for the assessment of the induced lymphocyte proliferation after the sensitization phase. For this purpose the mice were intravenously injected (into a tail vein) with radiolabelled thymidine (3H-methyl thymidine). Approximately five hours after the intravenous injection, the mice were sacrificed and the draining auricular lymph nodes excised and pooled per animal. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was then determined by the incorporation of 3H-methyl thymidine measured in a β-scintillation counter.

The second phase of the experiment (challenge phase) was performed after a recovery period during which possible irritation effects had subsided. Thus, on day 16 of the experiment, the remaining test item treated group was challenged with the same dose of the test item Isotridecyl methacrylate (25%) as used in the sensitization phase. The challenge control group was treated once with 25% Isotridecyl methacrylate and the other group received the vehicle only. Two days after the challenge application (on day 18) the lymphocyte proliferation of the individual animals of each group was assessed as described above.

In the standard LLNA, a test item is regarded as a sensitizer if the exposure to one or more test item concentration results in a 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). In the Challenge LLNA, a test item is regarded as a sensitizer if the challenge exposure to the test item results in an incorporation of 3HTdR that is distinctly higher than the primary response seen directly after the sensitization phase. As a further indication, the result of the additional test group (12.5%) was taken into account for interpretation of the test result (e.g. regarding a possible dose response).

The animals did not show signs of systemic toxicity during the course of the study and no cases of mortality were observed.

In the sensitization phase, a statistically significant increase was observed in the ear weights of the group treated with 25% test item concentration (day 1-3) in comparison to the vehicle control group and also to the group treated with 12.5% test item concentration (day 1-3) (p<0.001). In the challenge phase, a significant increase in ear weights was not observed in the group treated with 25% test item concentration (day 1-3 and day 16) or in the challenge control group (treated with the vehicle on day 1-3 and once with 25% test item concentration on day 16) in comparison to the control group. A single application of the test item in the challenge phase did thus not induce a relevant increase in ear weight or ear thickness. In contrast, with regard to the sensitization phase, the results of the ear

weight measurement were not reflected in the ear thickness measurements where no significant difference was seen between the ear thickness measured in test item treated animals and control animals. However, as the ear weight results are confirmed by the results obtained in the previous study with Isotridecyl methacrylate, they are considered to be more reliable.

In the sensitization phase of this study, Stimulation Indices (S.I.) of 1.35 and 3.06 were determined with the test item at concentrations of 12.5% and 25%, respectively. A statistically significant increase in DPM/animal was observed in the group treated with 25% test item concentration (day 1-3) in comparison to the vehicle control group (p=0.001). A statistically significant dose dependent increase was seen in this group in comparison to the group treated with 12.5% test item concentration (p=0.001).

After a single challenge application on day 16, an S.I. of 1.43 was obtained in the group treated with 25% test item concentration (day 1-3 and day 16). No statistically significant difference was seen between this group and the control group (treated with the vehicle on day 1-3 and on day 16) or the challenge control group (treated with the vehicle on day 1-3 and once with 25% test item concentration on day 16). The S.I. of the challenge control group was 1.42.

Thus, in the group treated with 25% test item concentration on day 1-3, a distinct lymphocyte proliferation was observed (S.I. = 3.06). However, as the response of animals challenged with 25% test item on day 16 (S.I. = 1.43) was lower and not distinctly higher than the primary response obtained with the same test item concentration directly after the sensitization phase, it can be concluded that the induced reponse does not bear any immunological memory. Thus, the lymphocyte proliferation observed in the sensitization phase is not due to a sensitizing effect but results from irritation caused by the test item as also supported by the ear weight data.

This is also supported by the fact that in the group treated with 12.5% test item concentration (day 1-3), where no relevant increase in ear weights (no irritation) was observed in comparison to the control group, the obtained S.I. was below the threshold of 3.

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Several studies are available to assess the sensitisation potential of Isotridecyl methacrylate. One GPMT test and two LLNA were conducted. While the GPMT study showed positive skin reactions, the first LLNA was considered to be inconclusive. Further evidence regarding the question whether the observed effect is truly of a sensitising nature was obtained in a follow-up LLNA with challenge. This latter study was clearly negative, which is also in line with supporting studies conducted withthe structurally related substancesMethacrylic acid ester 13.0andMethacrylic acid ester 17.4.

Overall, Isotridecyl methacrylate is not a sensitiser.

 

Guinea pig maximisation test

In a dermal sensitisation study according to OECD TG 406, 1992) with Isotridecyl methacrylate, young adult Dunkin-Hartley guinea pigs were tested using the Maximization test method.

Intradermal Induction was conducted with 25% w/v test item in arachis oil BP. For topical induction, the test item was used undiluted as supplied. Two challenge concentrations were used: undiluted as supplied and 75% v/v in arachis oil BP.

Very slight to well-defined erythema was noted at the intradermal induction sites of all test group animals at the 24 and 48-hour observations. Very slight erythema was noted at the intradermal induction sites of all control group animals at the 24-hour observation and in six control group animals at the 48-hour observation.

After topical induction, very slight to well-defined erythema, with or without very slight to slight oedema, were noted at the induction sites of all test group animals at the 1 and 24-hour observations. Bleeding from the intradermal induction sites was noted in fifteen test group animals at the 1-hour observation. Small superficial scattered scabs were noted at the induction sites of three test group animals at the 24-hour observation.

Very slight to well-defined erythema and an isolated incident of very slight oedema were noted at the treatment sites of all control group animals at the l-hour observation. Bleeding from the intradermal induction sites was noted at the treatment site of one control group animal. No skin reactions were noted at the treatment sites of control group animals at the 24-hour

observation.

After challenge with undiluted test item positive skin responses (very slight to well-defined erythema - grades 1 to 2) and incidents of very slight oedema were noted at the challenge sites of

seven test group animals at the 24-hour observation and in two test group animals at the 48-hour observation.

Challenge with 75% test item in Arachis Oil lead to positive skin responses (very slight to well-defined erythema - grades 1 to 2) and incidents of very slight oedema were noted at the challenge sites of four test group animals at the 24-hour observation and persisted in one test group animal at the 48-hour observation.

No skin reactions were noted at the challenge sites of control group animals at the 24 or 48-hour observations with either 100% or 75% test item concentration.

The positive control substance was 2-mercaptobenzothiazole with a sensitisation rate of 100 %.

In this study, the test item Isotridecyl methacrylate produced a 35% (7/20) sensitisation rate and was classified as a moderate sensitiser. 

 

Local lymph nose assays

In this study the test item Isotridecyl methacrylate dissolved in acetone:olive oil (4+1) was assessed for its possible contact allergenic potential using the Local Lymph Node Assay (LLNA) in mice in accordance with OECD Guideline 429 (2002). The principle of the LLNA is that sensitisers induce a primary proliferation of lymphocytes in the lymph node draining the application site. The ratio of proliferation in test item treated groups compared to that in vehicle controls is referred to as the Stimulation Index (S.I.). Radioactive labelling is used to measure cell proliferation. A test item is regarded as a sensitiser in the LLNA if exposure to one or more test item concentration results in a 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated test item concentration required to produce a S.I. of 3 is referred to as the EC3 value.

The main experiment was performed using test item concentrations of 25, 50, and 100%.

The animals did not show any signs of local irritation or systemic toxicity during the course of the study and no cases of mortality were observed.

In this study Stimulation Indices (S.I.) of 5.86, 5.52, and 6.83 were determined with the test item at concentrations of 25, 50, and 100% in acetone:olive oil (4+1), respectively. At all tested concentrations a statistically significant difference of DPM per animal was determined in comparison to the vehicle control group (p <0.001). Even though a clear dose-response was not obtained, all S.I. values were well above the threshold of 3.

However, a statistically significant and dose dependent increase in ear weights was obtained in all test item treated groups in comparison to the vehicle control group (p<0.001). Therefore, the potential skin irritating properties of the test item may have contributed to the observed increase in lymph node cell proliferation.

On the basis of the S.I. values, the test item Isotridecyl methacrylate would have to be classified as a skin sensitiser. But due to the unclear dose response relationship and the potentially irritating effects, a definite conclusion cannot be reached. Thus, the outcome of this study is considered to be inconclusive. Further evidence regarding the question whether the obtained effect is truly of a sensitising nature can be obtained using the LLNA with challenge.

 

The purpose of a follow-up Local Lymph Node Assay was to differentiate whether a lymphocyte proliferation observed in a previous study after treatment with Isotridecyl methacrylate was due to a true sensitizing effect or only due to irritation.

The principle of the assay is based on the immunological memory function of a sensitizing agent, which does not occur in irritation reactions.

Based on the results of the above mentioned study, 25% was chosen for evaluation in the Challenge-LLNA because at this concentration a lymphocyte proliferation above the threshold level of three was obtained without eliciting excessive irritation. 12.5% was included as an additional concentration for evaluation in the sensitization phase because it was expected that at this concentration, the test item would not have an irritative potential.

The present study was divided into 2 treatment phases, the sensitization phase and the challenge phase. Altogether, 6 groups each of five female mice were treated with the vehicle or the test item at different concentrations by topical application at the dorsum of each ear once daily each on three consecutive days:

A vehicle control group (acetone:olive oil (4+1)) for the sensitization phase, two test item groups for the sensitization phase treated with 12.5% and 25% test item concentration, a vehicle control group for the challenge phase which was treated with the vehicle only both in the sensitization phase and the challenge phase, a challenge control group which received vehicle only in the sensitization phase of the experiment and 25% test item in the challenge phase, and a test item group for the challenge phase treated with 25% test item concentration both in the sensitization phase and the challenge phase.

In the first phase (sensitization phase) of the study, on day 6 of the experiment the groups treated with test item concentrations of 12.5% and of 25% and one vehicle group were used for the assessment of the induced lymphocyte proliferation after the sensitization phase. For this purpose the mice were intravenously injected (into a tail vein) with radiolabelled thymidine (3H-methyl thymidine). Approximately five hours after the intravenous injection, the mice were sacrificed and the draining auricular lymph nodes excised and pooled per animal. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was then determined by the incorporation of 3H-methyl thymidine measured in a β-scintillation counter.

The second phase of the experiment (challenge phase) was performed after a recovery period during which possible irritation effects had subsided. Thus, on day 16 of the experiment, the remaining test item treated group was challenged with the same dose of the test item Isotridecyl methacrylate (25%) as used in the sensitization phase. The challenge control group was treated once with 25% Isotridecyl methacrylate and the other group received the vehicle only. Two days after the challenge application (on day 18) the lymphocyte proliferation of the individual animals of each group was assessed as described above.

In the standard LLNA, a test item is regarded as a sensitizer if the exposure to one or more test item concentration results in a 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). In the Challenge LLNA, a test item is regarded as a sensitizer if the challenge exposure to the test item results in an incorporation of 3HTdR that is distinctly higher than the primary response seen directly after the sensitization phase. As a further indication, the result of the additional test group (12.5%) was taken into account for interpretation of the test result (e.g. regarding a possible dose response).

The animals did not show signs of systemic toxicity during the course of the study and no cases of mortality were observed.

In the sensitization phase, a statistically significant increase was observed in the ear weights of the group treated with 25% test item concentration (day 1-3) in comparison to the vehicle control group and also to the group treated with 12.5% test item concentration (day 1-3) (p<0.001). In the challenge phase, a significant increase in ear weights was not observed in the group treated with 25% test item concentration (day 1-3 and day 16) or in the challenge control group (treated with the vehicle on day 1-3 and once with 25% test item concentration on day 16) in comparison to the control group. A single application of the test item in the challenge phase did thus not induce a relevant increase in ear weight or ear thickness. In contrast, with regard to the sensitization phase, the results of the ear

weight measurement were not reflected in the ear thickness measurements where no significant difference was seen between the ear thickness measured in test item treated animals and control animals. However, as the ear weight results are confirmed by the results obtained in the previous study with Isotridecyl methacrylate, they are considered to be more reliable.

In the sensitization phase of this study, Stimulation Indices (S.I.) of 1.35 and 3.06 were determined with the test item at concentrations of 12.5% and 25%, respectively. A statistically significant increase in DPM/animal was observed in the group treated with 25% test item concentration (day 1-3) in comparison to the vehicle control group (p=0.001). A statistically significant dose dependent increase was seen in this group in comparison to the group treated with 12.5% test item concentration (p=0.001).

After a single challenge application on day 16, an S.I. of 1.43 was obtained in the group treated with 25% test item concentration (day 1-3 and day 16). No statistically significant difference was seen between this group and the control group (treated with the vehicle on day 1-3 and on day 16) or the challenge control group (treated with the vehicle on day 1-3 and once with 25% test item concentration on day 16). The S.I. of the challenge control group was 1.42.

Thus, in the group treated with 25% test item concentration on day 1-3, a distinct lymphocyte proliferation was observed (S.I. = 3.06). However, as the response of animals challenged with 25% test item on day 16 (S.I. = 1.43) was lower and not distinctly higher than the primary response obtained with the same test item concentration directly after the sensitization phase, it can be concluded that the induced reponse does not bear any immunological memory. Thus, the lymphocyte proliferation observed in the sensitization phase is not due to a sensitizing effect but results from irritation caused by the test item as also supported by the ear weight data.

This is also supported by the fact that in the group treated with 12.5% test item concentration (day 1-3), where no relevant increase in ear weights (no irritation) was observed in comparison to the control group, the obtained S.I. was below the threshold of 3.

 

SupportingLocal lymph nose assays

In a dermal sensitization study with Methacrylic acid ester 13.0 dissolved in acetone:olive oil (4 +1) as a vehicle, 16 (4 per dose group) 8 -12 weeks old female CBA/CaOlaHsd mice were tested using the method of OECD 429 (Local Lmyphnode Assay). 

Three groups each of four female mice were treated daily with the test item at concentrations of 5%, 10 %, and 25 % (w/v) in acetone:olive oil (4 +1) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. A control group of four female mice was treated with the vehicle (acetone:olive oil (4 +1)) only.

In the course of the study no cases of mortality were observed. Systemic signs of toxicity were not observed during the experiment. Local effects (ear reddening) were only observed with the highest treatment dose (25%) 24 hours after the second and 1 hour after the third application but not on day 6.

In this study Stimulation Indices (S.I.) of 0.99, 2.11 and 2.66 were determined with the test item at concentrations of 5, 10, and 25% in acetone:olive oil (4 +1), respectively. No dose-response relationship was observed. The EC3 value could not be determined since none of the tested concentrations induced an S.I. value greater than 3.

In this study, Methacrylic acid ester 13.0 is therefore not a dermal skin sensitizer under the described conditions.

 

In order to study a possible contact allergenic potential of Methacrylic acid ester 17.4, three groups each of four female mice were treated daily with the test item at concentrations of 2.5 %, 5 % and 10 %(w/v) in acetone/olive oil (4/1, v/v) by topical application 10 the dorsum of each ear lobe (Ieft and right) for three consecutive days. A control group of four female mice was treated with the vehicle acetone/olive oil (4/1, vIv) only.

Five days after the first topical application the mice were injected intravenously into a tail vein with radio-Iabelled thymidine (3H-methyl thymidine, 3HTdR). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of Iymph node cells were prepared from pooled Iymph nodes which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled Iymph node cells was determined by the incorporation of 3HTdR measured in a ß-scintillation counter.

All treated animals survived the scheduled study period.

Neither clinical / local signs nor other findings were observed in any animals of the control group, Group 2 (2.5 %) or Group 3 (5 %). One day after the first topical application, slight ear erythema was observed at both dosing sites in all mice of Group 4 (10 %) persisting for a total of two days.

S.I. values of 0.9, 0.9 and 1.9 were obtained for the groups treated with 2.5, 5 and 10% test item, respectively.

No dose-response relationship was observed. An EC3 value could not be determined because all SI values were below 3. Thus, based on the results of this study, Methacrylic acid ester 17.4 is not a sensitiser.

 

 

There are no data gaps for the endpoint skin sensitisation. No human information is available for this endpoint. However, there is no reason to believe that the available results would not be applicable to humans.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

There is no information available for respiratory sensitisation. Therefore, there is a data gap in this respect. However, the data gap cannot be filled with experimental data, since there is no internationally accepted animal model for respiratory sensitisation. In case human data for respiratory sensitisation emerges, this will be taken into account. For skin sensitisation, there is no reason to believe that results obtained in guinea pigs would not be applicable to humans.

Justification for classification or non-classification

Based on the available data, Isotridecyl methacrylate does not need to be classified for skin sensitisation according to regulation (EC) 1272/2008. Thus, no labelling is required.