Reaction mass of 2,2'-[methylenebis(oxymethylene)]bis[2-ethylpropane-1,3-diol]and propylidynetrimethanol and 2,2'-[oxybis(methylene)]bis[2-ethylpropane-1,3-diol]

Endpoint summary

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant and conducted in accordance with testing guidelines.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/C

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Manston Road, Margate, Kent, UK.
- Age at study initiation: 8 - 11 weeks old
- Weight at study initiation: 15.7 - 20.3g
- Housing: All mice were housed in pairs in solid-bottomed polypropylene cages with a stainless steel grid top and an integrated food hopper. Wood shavings were used as bedding and nesting material (‘Nestlets’) was provided.
- Diet (e.g. ad libitum): SDS Rat and Mouse (modified) No. 1 Diet SQC Expanded was available ad libitum.
- Water (e.g. ad libitum): Water taken from the public supply was available from water bottles ad libitum throughout the study.
- Acclimation period: At least 8 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The average daily environmental temperature was approximately 21°C
- Humidity (%): 45% to 51%.
- Air changes (per hr): Minimum of 10 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 h light/dark cycle was in operation

IN-LIFE DATES: From: To:

Vehicle:

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

Concentration:

Preliminary study: 50% concentration used.
Main study: 10, 25 and 50% concentrations were used.

No. of animals per dose:

2 females in the preliminary study.
4 females per dose group were used in the main study.

Details on study design:

RANGE FINDING TESTS:
Preliminary testing was conducted. Two female mice received an open application of 25 μL of test item formulation onto the dorsum of each ear once daily on 3 consecutive days (Days 1 to 3), with no further treatment after the 3rd application. The animals were checked for viability twice a day and were frequently observed for signs of reaction to treatment on each day of dosing (pre-dose, immediately post dose and 1 and 2 hours after dosing) and once daily thereafter. The body weight of each individual animal was recorded on Day 1 (before the first dose) and on Day 6. On Day 1 (pre-dose) and on Day 3 (approximately 48 h after the first dose) and Day 6 the thickness of both ears of each mouse was measured using digital callipers. In addition, both ears were observed for erythema and the scores recorded.

Animals were euthanised on Day 6 and were then discarded. There were no signs of systemic toxicity and no effect on body weight was noted. Between Days 1 and 3 both mice recorded unilateral increases in ear thickness of ≥25%. However, no erythema was recorded in either mouse during the observation period.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Mouse Local Lymph Node Assay.
- Criteria used to consider a positive response:

TREATMENT PREPARATION AND ADMINISTRATION:
Formulations were prepared on each day of dosing. The test item was placed in a sealed bag and immersed in water that was heated to a maximum temperatureof 80°C until it was visibly homogeneous. The required amount of HB TMP was weighed into a pre-labelled container and formulations were made up to the final volume by adding the requisite amount of AOO. Formulations were stirred using a magnetic stirrer.

Treatment was administered on 3 consecutive days (Days 1 to 3). On each day of treatment the animals received an open application of 25 μL of the appropriate formulation onto the dorsum of each ear.

On Day 6 each animal received an intravenous injection (250 μL) of phosphate buffered saline (PBS) containing 19.3 μCi of [methyl-3H] thymidine into the lateral tail vein. Approximately 5 h after intravenous administration all animals were euthanised by exposure to a rising concentration of carbon dioxide and the major blood vessels were severed to exsanguinate. Each pair of draining auricular lymph nodes was collected from each animal and the animal was then discarded. A single cell suspension of lymph node cells from each paired sample was prepared by gentle disaggregation through a 200 μm mesh stainless steel gauze into conical centrifuge tubes. The lymph node cells were washed in an excess of PBS (approximately 1 mL) and the mesh was discarded. The lymph node cells were then centrifuged at approximately 1300 g for 10 min at 4°C. The supernatant was drawn off and the cells were washed a second time with approximately 1 mL PBS and then centrifuged (also at approximately 1300 g for 10 min at 4°C). Following centrifugation, the supernatant was discarded and the DNA was precipitated with approximately 1 mL 5% trichloroacetic acid at 2 to 8°C for approximately 18½ h. The resulting pellet was again centrifuged at approximately 1300 g for 10 min at 4°C and the supernatant discarded. The pellet was then re-suspended in 200 μL ‘Solvable’, an aqueous-based solubiliser, and the suspension transferred to a vial containing 10 mL scintillation fluid. Incorporation of tritiated thymidine was measured by β- scintillation counting and was expressed as disintegrations per minute (DPM).

Positive control substance(s):

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

Statistics:

Group means and standard deviations were calculated for disintegrations per minute. Group means and standard deviations were calculated for body weights, along with body weight gains. The percentage change in ear thickness of preliminary test mice was calculated. Dixon’s Q-test for the detection of a single outlier was performed on disintegrations per minute values. No other formal statistical analysis was carried out.

Positive control results:

No information provided.

Parameter:

SI

Value:

1.1

Test group / Remarks:

Group 1 (25%)

Remarks on result:

other: The stimulation indices for mice treated with the test item at concentrations of 10%, 25% and 50%, when compared with the control group, were 1.1, 1.4 and 1.5, respectively.

Parameter:

SI

Value:

1.4

Test group / Remarks:

Group 2 (50%)

Remarks on result:

other:

Parameter:

SI

Value:

1.5

Test group / Remarks:

Group 3 (100%)

Remarks on result:

other:

There were no systemic signs and no signs of local irritation in any animal during the observation period. Clinical signs were restricted to wetness to the head, which was observed in all animals at the 1 h and 2 h post dose observations on Days 1 and 2. Among mice treated with the 10% concentration this sign persisted until Day 3 and among mice treated with the 25% concentration and those treated with the 50% it persisted until Day 4. However, this sign was considered to be merely an accumulation of formulation residues.

All mice gained weight during the observation period and these gains were considered to be acceptable for mice of this age and strain.

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study, treatment with HB TMP up to concentrations of 50% did not achieve a stimulation index of ≥3 and as a

Executive summary:

A study conducted in accordance with OECD Guideline 429 was performed to determine the ability of HB TMP to cause skin sensitisation in a mouse LLNA study using female CBA/Ca mice. A preliminary study was conducted initially, using two mice and a concentration of 50% HB TMP and vehicle (acetone:olive oil, 4:1 v/v). Both mice received an open application of 25 μL of test formulation onto the dorsum of each ear on 3 consecutive days. Both mice survived and there were no signs of systemic toxicity or excessive local skin irritation.

In the main study, three formulation concentrations of 10%, 25% and 50% in vehicle were tested, with four mice per test group. A vehicle control group was also included. Each mouse received an open application of 25 μL of test formulation onto the dorsum of each ear. Animals were treated on 3 consecutive days. Three days after the final application each animal received an intravenous injection of [methyl-3H] thymidine into the lateral tail vein. Approximately 5 h later the draining lymph nodes were collected in order that incorporation of tritiated thymidine could be assessed by scintillation counting.

Clinical observations for signs of toxic reaction, mortality and moribundity and body weights were measured.

There were no signs of systemic toxicity and body weight gains were considered to be acceptable for mice of this age and strain.

The stimulation indices (SI) for Groups 2 (25% dose group), 3 (50% dose group) and 4 (100% dose group), when compared with Group 1 (control), were 1.1, 1.4 and 1.5, respectively.

Under the conditions of the study, since treatment with HB TMP at concentrations of up to 50% did not achieve a stimulation index of ≥3, it was considered that the test item does not have the potential to cause skin sensitisation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The skin sensitisation potential of the Reaction mass of 2,2'-[methylenebis(oxymethylene)]bis[2-ethylpropane-1,3-diol] and 2,2'-[oxybis(methylene)]bis[2-ethylpropane-1,3-diol] and propylidynetrimethanol was investigated in a skin sensitisation study conducted according to OECD Test Guideline 429 using the local lymph node assay (Robertson, 2012). In the study, four groups of female mice were treated with either control or the test substance, HB TMP, at concentrations of 25, 50 and 100%. The mice received open applications of 25 μL of test formulation onto the dorsum of each ear, with the control receiving vehicle only (acetone:olive oil). Treatments were administered on three consecutive days. On the 6th day, the test animals were euthanized. The test animals were checked twice daily for viability, with body weights recorded on day 1 (prior to testing) and on day 6 and all animals were examined for their reactions to treatment. No signs of systemic toxicity were observed in treated animals and body weight gains were considered to be acceptable for mice of this age and strain.

The stimulation indices (SI) for doses of HB TMP of 25%, 50% or 100% when compared with controls, were 1.1, 1.4 and 1.5, respectively. Under the conditions of this study, treatment with HB TMP at concentrations of up to 100% did not cause skin sensitisation. On this basis, the Reaction mass of 2,2'-[methylenebis(oxymethylene)]bis[2-ethylpropane-1,3-diol] and 2,2'-[oxybis(methylene)]bis[2-ethylpropane-1,3-diol] and propylidynetrimethanol is not a skin sensitiser.

Migrated from Short description of key information:
The reaction mass of 2,2'-[methylenebis(oxymethylene)]bis[2-ethylpropane-1,3-diol] and 2,2'-[oxybis(methylene)]bis[2-ethylpropane-1,3-diol] and propylidynetrimethanol was not found to cause skin sensitisation in a study conducted using the local lymph node assay. There is no evidence from experience of use that the substance has the potential to cause skin or respiratory sensitisation in exposed workers.

Justification for selection of skin sensitisation endpoint:
Sole study providing data from a guideline compliant study. Under the conditions of the study, the test substance was not found to be a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

There is no indication from the experience of use that the Reaction mass of 2,2'-[methylenebis(oxymethylene)]bis[2-ethylpropane-1,3-diol] and 2,2'-[oxybis(methylene)]bis[2-ethylpropane-1,3-diol] and propylidynetrimethanol can cause respiratory sensitisation in exposed workers.

Migrated from Short description of key information:
There is no indication from reports in exposed workers that the substance can cause respiratory sensitisation (occupational asthma).

Justification for classification or non-classification

The Reaction mass of 2,2'-[methylenebis(oxymethylene)]bis[2-ethylpropane-1,3-diol] and 2,2'-[oxybis(methylene)]bis[2-ethylpropane-1,3-diol] and propylidynetrimethanol did not cause skin sensitisation in a study conducted using the local lymph node assay. The substance does not meet the criteria for classification for skin or respiratory sensitisation according to Directive 67/548/EEC or Regulation 1272/2008/EC.