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Description of key information

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:
7 to 26 October 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Proprietary GLP guideline-compliant study.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Twenty-two female (nulliparous and non-pregnant) mice of the CBA/Ca strain were used. All animals were supplied by Charles River UK Limited, Manston Road, Margate, Kent, UK and arrived at Charles River, Edinburgh on 29 September 2009. They were 7 to 8 weeks old and weighed 16 to 20 g on despatch. The mice were acclimatised for at least 8 days, and were randomly assigned to treatment groups on arrival. Each animal received a subcutaneous implant which identified it individually within the study and which corresponded to that animal's number.
The animals were housed in groups of 2 or 3 in polycarbonate cages (dimensions 36.5 x 20.7 x 14 cm) with a stainless steel grid top and an integrated food hopper. Wood shavings were used as bedding and nesting material (‘Nestlets’) was provided. Both were supplied by Datesand Limited, Manchester, UK. A wooden chewstick, supplied by Estap OÜ, 75401 Harjumaa, Estonia, was placed in each cage as environmental enrichment. Certificates of analysis for bedding, Nestlets and chewsticks were retained by the laboratory. Analysis did not provide evidence of contamination that might have prejudiced the outcome of the study. Each cage was supplied with a water bottle.
The environment is monitored throughout the day and recordings are made every 15 min. From animal arrival to the end of the observation period, average daily environmental temperatures were within a range of approximately 21 to 22oC and the range for average daily relative humidity was approximately 46 to 61%. A 12 h light/dark cycle was in operation (light hours 0700 to 1900 h) with a minimum of 15 air changes per hour.
Rat and Mouse No. 1 Maintenance Diet (Special Diets Services, PO Box 705, Witham, Essex, UK) and water taken from the public supply (Scottish Water, Edinburgh, Midlothian, UK) were available ad libitum throughout the study. Each batch of diet is routinely analysed by the supplier for various nutritional components and chemical and microbiological contaminants. The quality of water supply is stipulated by legislation in Water Quality, Scotland, Regulations 2001 and certificates of analysis for dissolved materials, heavy metals, pesticide residues, pH, nitrates and nitrites are periodically provided. These analyses are based on water samples taken from these laboratories. The results of diet and water analyses did not provide evidence of contamination and so the outcome of the study was not prejudiced.
Vehicle:
propylene glycol
Concentration:
0, 10, 25 and 50%
No. of animals per dose:
5 per/concentration
Details on study design:
Preliminary test: 2 females were treated with 25 µL of the 50% concentration on the dorsum of each ear for 3 consecutive days (Days 1-3). There was no treatment on Days 4 and 5. Observations were conducted frequently on each day of dosing (predose, immediately post dose and approximately 1 and 2 h after dosing) and once daily thereafter until the kill, by an intravenous overdose of sodium pentobarbitone, on Day 6. The animals were then discarded.There were no signs of systemic toxicity or local irritation and no effect on body weight was noted. Therefore, dose concentrations of 10%, 25% and 50% were selected as suitable non-toxic doses for administration in the main study.

Main study: For 3 consecutive days (Days 1 to 3) animals received an open application of 25 μL of the appropriate formulation onto the dorsum of each ear. There was no treatment on Days 4 and 5. On Day 6 each animal received an intravenous injection (250 μL) of phosphate buffered saline (PBS) containing 18.7 μCi of [methyl-3H] thymidine into the lateral tail vein. Approximately 5 h after intravenous administration all animals were killed by exposure to a rising concentration of carbon dioxide and the major blood vessels were severed to exsanguinate. The lymph nodes were collected according to the methods below.
All animals were checked for viability early in the morning and again as late as possible on each day. On each day of dosing the observations were conducted predose, immediately post dose and approximately 1 and 2 h post dose. Thereafter animals were observed once daily until kill on Day 6 (the d ay of the thymidine injection). The body weight of each individual animal was recorded on Day 1 (before the first dose) and on Day 6.

Formulations of Bis-MPA were prepared on each day of dosing. A correction factor of 1.02 was used in preparing each formulation in order to take account of the test item purity. For each formulation the appropriate amount of Bis-MPA was weighed and transferred to a suitable glass container. Propylene glycol was added and the mixture was stirred using a magnetic stirrer and either a mechanical mixer or a mini-homogeniser until the formulation was visibly homogeneous. The formulation was then made up to the final volume. An appropriate amount of propylene glycol was also dispensed for administration to the vehicle control group. The pH of the formulation prepared on Day 1 of the preliminary test and also of each of the formulations prepared on Day 1 of the main study was pH 7.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
No formal statistical analysis was carried out.
Positive control results:
The stimulation indices in a recent positive control study were 1.7, 2.4 and 5.0 for 5%, 10% and 25% hexylcinnamicaldehyde, respectively.
Parameter:
SI
Remarks on result:
other: The stimulation indices for mice treated with the test item at concentrations of 10%, 25% or 50%, when compared with the control group, were 1.5, 1.2 and 1.2, respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: DPM for mice (group means) treated with the test item at concentrations of 0%, 10%, 25% or 50% were 1733, 2610, 2082 and 2131, respectively.

No systemic signs were noted in any animal during the observation period. Body weight 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 the study, since treatment with Bis-MPA 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 sensitisation.
Executive summary:

This LLNA study investigated the delayed contact hypersensitivity potential of the test item, Bis-MPA, using CBA/Ca mice. A preliminary test was conducted using 2 females. Each mouse received an open application of 25 μL of a 50% formulation of Bis-MPA onto the dorsum of each ear on 3 consecutive days. There was no evidence of systemic toxicity or local irritation and, as a result of these findings, formulation concentrations were selected for the main study. Three groups, each consisting of 5 females, were treated in the same manner as the preliminary test mice with concentrations prepared at 10%, 25% and 50%, respectively, also for 3 consecutive days. The vehicle was propylene glycol and one group of 5 females received only this and acted as controls. Three days after the final application each animal received an intravenous injection of [methyl-3H] thymidine into the lateral tail vein and 5 h later the draining lymph nodes were collected in order that the incorporation of tritiated thymidine could be assessed by scintillation counting.

There were no systemic signs noted in any animal during the observation period and body weight changes were considered to be acceptable for mice of this age and strain. The stimulation index (SI) values for the mice treated with Bis-MPA at concentrations of 10%, 25% or 50%, when compared with the control group, were 1.5, 1.2 and 1.2, respectively.

Under the conditions of the study, since treatment with Bis-MPA 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 sensitisation. Bis-MPA did not meet the criteria for classification as a sensitiser according to EU labelling regulations Commission Directive 2001/59/EC. Hence, there is no requirement for either a symbol or a risk phrase. According to the criteria in the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) (Amendments to the second revised edition, ST/SG/AC.10/30/Rev.2) Bis-MPA is not classified as a skin sensitiser.

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

The delayed contact hypersensitivity potential of Bis-MPA (dimethylol propionic acid) was investigated in a GLP LLNA conducted according to OECD Guideline 429 (Robinson, 2010). A preliminary test was conducted using 2 female CBA/Ca mice.

Each mouse received an open application of 25 μL of a 50% formulation of Bis-MPA onto the dorsum of each ear on 3 consecutive days. There was no evidence of systemic toxicity or local irritation and, as a result of these findings, formulation concentrations were selected for the main study. Three groups, each consisting of 5 females, were treated in the same manner as the preliminary test mice with concentrations prepared at 10%, 25% and 50%, respectively, also for 3 consecutive days. There were no systemic signs noted in any animal during the observation period and body weight changes were considered to be acceptable for mice of this age and strain. The stimulation index (SI) values for the mice treated with Bis-MPA at concentrations of 10%, 25% or 50%, when compared with the control group, were 1.5, 1.2 and 1.2, respectively. Under the conditions of the study, since treatment with Bis-MPA 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 sensitisation.


Migrated from Short description of key information:
A modern, guideline-compliant LLNA shows that Bis-MPA (dimethylol propionic acid) does not have the potential to cause skin sensitisation.

Justification for selection of skin sensitisation endpoint:
Only study available for this endpoint

Justification for classification or non-classification

Dimethylol propionic acid did not meet the criteria for classification as a sensitiser according to EU labelling regulations Commission Directive 2001/59/EC. Hence, there is no requirement for either a symbol or a risk phrase. According to the criteria in the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) (Amendments to the second revised edition, ST/SG/AC.10/30/Rev.2) Dimethylol propionic acid is not classified as a skin sensitiser.