Reaction mass of dodecyl methacrylate and tridecyl methacrylate

Endpoint summary

Description of key information

not sensitising (LLNA, OECD TG 429, RL1, GLP): SI: 1.38, 1.95, and 2.45 at concentrations of 5%, 25%, and 50% (v/v) test item in acetone:olive oil (4:1 v/v)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 December 2018 to 02 January 2019

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:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Animal Breeding Facility, Jai Research Foundation
- Age at study initiation: 11- 12 weeks
- Weight at study initiation: Minimum: 20.7, Maximum: 27.4
- Housing: On the days of test item application (Days 0, 1 and 2), the animals were housed in individual cages. From day 3 the animals were group housed 5 mice/cage.
- Diet (e.g. ad libitum): Teklad Certified Global High Fiber Rat/Mice Feed manufactured by Envigo, USA, ad libitum
- Water (e.g. ad libitum): UV sterilised water (Reverse Osmosis water filtration system), ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 23 °C
- Humidity (%): 58 to 66%
- Air changes (per hr): Minimum 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

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

Concentration:

5, 25, 50% based on preliminary assay: In the preliminary assay, no erythema was observed at tested concentrations of 10% (v/v), 50% (v/v), 75% (v/v) and 100%, however an increase in ear thickness of more than 25% was observed at tested concentrations of 75% (v/v) and 100%.

No. of animals per dose:

prelim. study: 2/group
main study: 5/group

Details on study design:

PRE-SCREEN TESTS:
- concentrations tested: 10% (v/v), 50% (v/v), 75% (v/v) in acetone:olive oil (4:1 v/v) and 100% (25 μL/ear)
- Compound solubility: The test item was soluble in acetone:olive oil (4:1 v/v) (up to 75%).
- Irritation: No erythema was observed at tested concentrations of 10% (v/v), 50% (v/v), 75% (v/v) and 100%, however an increase in ear thickness of more than 25% was observed at tested concentrations of 75% (v/v) and 100%.
- Systemic toxicity: no

MAIN STUDY
Three groups (G2 to G4) were treated topically for three consecutive days (days 0, 1 and 2) on the dorsal surface of both ears (25 µL/ear) at concentrations of 5%, 25% and 50% (v/v) test item in acetone:olive oil (4:1 v/v), respectively. Mice from the vehicle control group (G1) and positive control group (G5) were handled in the same manner but received 25 L/ear of acetone:olive oil (4:1 v/v) and 25% (v/v) alpha-Hexylcinnamaldehyde in acetone:olive oil (4:1 v/v), respectively.

TREATMENT
- Criteria used to consider a positive response:
The DPM/mouse, along with an appropriate measure of inter-animal variability (i.e., mean ± standard deviation), were calculated for each test group and vehicle and positive control groups. Final results were expressed as the Stimulation Index (SI) which is calculated as a ratio of the mean DPM of the test group divided by mean DPM of the vehicle control group. If the test item produces a SI  3 in the LLNA, it is considered positive for contact sensitisation potential and therefore an EC3 is determined. EC3 value (theoretical concentration resulting in a SI value of 3) is determined by linear interpolation of points on the dose-response curve, immediately above and below the 3-fold threshold (Basketter et al., 1999). The equation used for calculation of EC3 was:
EC3 = c + [(3 - d)/(b - d)] x (a - c)
Where a = the lowest concentration giving stimulation index > 3; b = the actual stimulation index caused by a; c = the highest concentration failing to produce a stimulation index of 3; and d = the actual stimulation index caused by c.
If the test item produces a SI < 3 in the LLNA, it is considered negative for contact sensitisation potential and therefore an EC3 is not determined. If EC3 value is found to be ≤ 2%, the test item is classified under Sub-category 1A, while if EC3 value is found to be > 2% it is classified under sub-category 1B based on the Globally Harmonized System of Classification and Labelling of Chemicals (GHS 2017).

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

In addition to an assessment of the magnitude of the SI, statistical analysis was carried out for the assessment of the dose response relationship and a pair-wise comparison made between the
treatment and the solvent/vehicle control group. All parameters characterised by continuous data such as body weight and radioactive disintegrations per minute (DPM) were subjected to Bartlett’s
test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA). To compare vehicle and positive control data, a Student’s t-test was performed to calculate significance.

Positive control results:

The SI of 6.12 obtained for the positive control, -Hexylcinnamaldehyde, showed a greater than three-fold increase over the control value indicating a positive response in agreement with the historical control for this known weak sensitiser. This confirmed the reliability of this test procedure.

Parameter:

SI

Value:

1.38

Test group / Remarks:

5% (v/v) test item in acetone:olive oil (4:1 v/v)

Parameter:

SI

Value:

1.95

Test group / Remarks:

25% (v/v) test item in acetone:olive oil (4:1 v/v)

Parameter:

SI

Value:

2.45

Test group / Remarks:

50% (v/v) test item in acetone:olive oil (4:1 v/v)

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
Proliferative responses in the draining lymph nodes were monitored by measuring the incorporation of 3H-methyl thymidine. These analyses revealed group mean DPM values of 931.80, 1288.40, 1816.60, and 2283.00 for the vehicle control, 5%, 25%, and 50% (v/v) test item in acetone:olive oil (4:1 v/v), respectively. The DPM value for positive control (25% alpha-Hexylcinnamaldehyde) was 5705.40.
A statistically significant increase in the mean DPM was observed in the 25% (v/v) and 50% (v/v) treatment groups and the 25% (v/v) HCA when compared to vehicle control group values.

DETAILS ON STIMULATION INDEX CALCULATION
Stimulation Index (SI) values calculated for groups treated with test item were 1.38, 1.95, and 2.45 at dose concentrations of 5%, 25%, and 50% (v/v) in acetone:olive oil (4:1 v/v), respectively, and 6.12 for the 25% v/v HCA treated positive control group.

EC3 CALCULATION
Since the SI obtained for the test item at all tested concentrations showed a less than three-fold increase over the vehicle control value; an EC3 calculation was not calculated.

CLINICAL OBSERVATIONS:
No clinical signs were observed in any mouse from the vehicle control, positive control, or any treated group at 5%, 25% and 50% (v/v) test item in acetone:olive oil (4:1 v/v). No erythema was observed in any treated mouse at 5%, 25%, and 50% (v/v) test item in acetone:olive oil (4:1 v/v) on day 0 to day 5. Very slight erythema was observed in the group treated with 25% HCA (during days 1 to 4) in all mice (5/5 mice).

BODY WEIGHTS
The mean body weight of positive control and test item treated mice was comparable to that of the vehicle control group.

Interpretation of results:

GHS criteria not met

Conclusions:

A Stimulation Index (SI) value of three or more (SI value of the treated group over the control group) indicates potential to cause skin sensitisation. The SI obtained for the test item at all tested concentrations showed a less than three-fold increase over the control value. Therefore, the test item did not demonstrate dermal sensitisation potential in the local lymph node assay.

Executive summary:

The Local Lymph Node Assay was conducted to evaluate the skin sensitisation potential of Reaction mass of dodecyl methacrylate and tridecyl methacrylate in CBA/J strain mice, in compliance with OECD test guideline 429 and test method EC B.42.

A preliminary assay was conducted to identify the appropriate test concentrations for use in the main Local Lymph Node Assay.

In the main assay, five groups of mice comprising 5 females/per group were selected. Based on results of the preliminary assay, three groups were treated at dose concentrations of 5%, 25%, and 50% (v/v) test item in acetone: olive oil (4:1; v/v) (AOO) for three consecutive days (days 0, 1 and 2) on the dorsum of both ears (25 µL per ear). In addition, one group served as the vehicle control and was treated with acetone:olive oil (4:1 v/v) and another group served as the positive control and was treated at 25% (v/v) HCA (alpha-hexylcinnamaldehyde) in acetone:olive oil (4:1 v/v). Mice were observed for clinical reactions. Mice were weighed at the beginning and the end of treatment.

On day 5, the uptake of intravenously injected 3H-methyl thymidine into the auricular (local) lymph nodes draining at the site of chemical application was measured (5 hours post-administration) to assess the lymph node proliferative response.

There was no indication of local irritation or systemic toxicity in test item treated mice.

In all mice treated with 25% (v/v) HCA, a local reaction consisting of very slight erythema (score of 1) was observed from days 1 to 4. The group mean body weight of test item treated mice and positive

control group mice were comparable with the mean body weight of the vehicle control group.

Stimulation indices (SI) for the 5%, 25%, and 50% (v/v) test item in acetone:olive oil (4:1 v/v) treated groups were 1.38, 1.95, and 2.45, respectively (i.e., less than three-fold increase over mean vehicle control group). Therefore, the test item did not demonstrate dermal sensitisation potential in the local lymph node assay. All criteria for a valid study were met. The vehicle

control and positive control in the definitive LLNA were within acceptable ranges and fulfilled requirements for a valid assay.

A positive response for HCA (SI = 6.12) confirmed the reliability of the test procedure.

Based on results of this study, the test item is not classified as skin sensitiser.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The Local Lymph Node Assay was conducted to evaluate the skin sensitisation potential of Reaction mass of dodecyl methacrylate and tridecyl methacrylate in CBA/J strain mice, in compliance with OECD test guideline 429 and test method EC B.42.

A preliminary assay was conducted to identify the appropriate test concentrations for use in the mainLocal Lymph Node Assay.

In the main assay, five groups of mice comprising 5 females/per group were selected. Based on results of the preliminary assay, three groups were treated at dose concentrations of 5%, 25%, and 50% (v/v) test item in acetone: olive oil (4:1; v/v) (AOO) for three consecutive days (days 0, 1 and 2) on the dorsum of both ears (25 µL per ear). In addition, one group served as the vehicle control and was treated with acetone:olive oil (4:1 v/v) and another group served as the positive control and was treated at 25% (v/v) HCA (alpha-hexylcinnamaldehyde) in acetone:olive oil (4:1 v/v). Mice were observed for clinical reactions. Mice were weighed at the beginning and the end of treatment.

On day 5, the uptake of intravenously injected 3H-methyl thymidine into the auricular (local) lymph nodes draining at the site of chemical application was measured (5 hours post-administration) to assess the lymph node proliferative response.

There was no indication of local irritation or systemic toxicity in test item treated mice.

In all mice treated with 25% (v/v) HCA, a local reaction consisting of very slight erythema (score of 1) was observed from days 1 to 4. The group mean body weight of test item treated mice and positive

control group mice were comparable with the mean body weight of the vehicle control group.

Stimulation indices (SI) for the 5%, 25%, and 50% (v/v) test item in acetone:olive oil (4:1 v/v) treated groups were 1.38, 1.95, and 2.45, respectively (i.e., less than three-fold increase over mean vehicle control group). Therefore, the test item did not demonstrate dermal sensitisation potential in the local lymph node assay. All criteria for a valid study were met. The vehicle

control and positive control in the definitive LLNA were within acceptable ranges and fulfilled requirements for a valid assay.

A positive response for HCA (SI = 6.12) confirmed the reliability of the test procedure.

Based on results of this study, the test item is not classified as skin sensitiser.

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, Reaction mass of dodecyl methacrylate and tridecyl methacrylate does not need to be classified for skin sensitisation according to regulation (EC) 1272/2008. Thus, no labelling is required.