Octyl (R)-2-(4-chloro-2-methylphenoxy)propionate

Skin sensitisation

Administrative data

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 guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2010) - modified LLNA (IMDS): Measurement of cell counts instead of radioactive labeling. In addition, measurements of ear swelling and ear weights were done to discriminate the irritating potential from the sensitizing potential of the test substance.

Principles of method if other than guideline:

Modified LLNA (IMDS; Integrated Model for the Differentiation of Skin Reactions). Modifications are authorized in the OECD TG 429 and in the Note for Guidance SWP/2145/00 of the CPMP (2001). Information on validation of IMDS and scientific justification is given in: Vohr HW et al., Arch. Toxicol., 73, 501-509 (2000); Ehling G et al., Toxicology 212, 60-68 and 69-79 (2005).

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Octyl (R)-2-(4-chloro-2-methylphenoxy)propionate

EC Number:

266-358-7

EC Name:

Octyl (R)-2-(4-chloro-2-methylphenoxy)propionate

Cas Number:

66423-13-0

Molecular formula:

C18H27ClO3

IUPAC Name:

octyl (2R)-2-(4-chloro-2-methylphenoxy)propanoate

Constituent 2

Reference substance name:

Mecoprop-P n-octyl ester

IUPAC Name:

Mecoprop-P n-octyl ester

Constituent 3

Reference substance name:

Preventol B5

IUPAC Name:

Preventol B5

Constituent 4

Reference substance name:

R-(+)-2-(4-chloro-2-methylphenoxy)-propionic acid, octyl ester

IUPAC Name:

R-(+)-2-(4-chloro-2-methylphenoxy)-propionic acid, octyl ester

In vivo test system

Test animals

Species:

mouse

Strain:

NMRI

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Strain: Hsd Win: CRL: NMRI BR (SPF-bred)
- Source: Charles River Germany Sulzfeld, 97633 Sulzfeld, Germany
- Age at study initiation: 7 weeks
- Weight at study initiation: 26-32 g
- Housing: During the study period the animals were single-housed in Makrolon type II cages.
- Diet and water: ad libitum
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2° C
- Humidity (%): 40%-70%
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

0 (vehicle control), 10, 30, 100 %

No. of animals per dose:

6

Details on study design:

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was formulated once at day 1 of the study in acetone/olive oil. The formulation was applied epicutaneously onto the dorsal part of both ears of the animals. This treatment was repeated on three consecutive days (d1, d2 and d3). The volume administered was 25 µL/ear. The concentrations used were based on the experiences with the test system and the toxic properties of the test substance. For negative control a dose group treated only with the vehicle acetone /olive oil in the above described manner was used.
The animals were anaesthetized by inhalation of carbon dioxide and sacrificed one day after the last application (d4). The appropriate organs were then removed. Lymphatic organs (the auricular lymph nodes) were transferred into physiological saline (PBS).
Investigations:
- weight of lymph nodes
- cell counts in lymph nodes
- stimulation index (calculated by dividing the absolute number of weight or cell counts of the substance treated lymph nodes by the vehicle treated ones)
- ear swelling
- ear weight
- body weights

Positive control substance(s):

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

Statistics:

When it was statistically reasonable, the values from treated groups were compared with those from the control group by a one-way analysis of variance (ANOVA) when the variances are considered homogeneous according to a homogeneity testing like Cochran's test. Alternatively, if the variances are considered to be heterogeneous (p<=0.05), a non-parametric Kruskal-Wallis test has been used (Kruskal-Wallis ANOVA) at significance levels of 5%. Two sided multiple test procedures were done according to Dunnett or Bonferroni-Holm, respectively. Outlying values in the LN weights were eliminated at a probability level of 99% by Nalimov's method. In addition, for the LLNA/IMDS the smallest significant differences in the means were calculated by Scheffels method, which according to Sachs can be used for both equal and unequal sample sizes.

Results and discussion

Positive control results:

Alpha hexyl cinnamic aldehyde shows a clear increase for cell counts compared to control animals, which is of statistical significance. The “positive level”, which is 1.4 for cell count indices, has clearly been exceeded.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

Compared to vehicle treated animals there was a clear increase regarding cell counts, which is of statistical significance in the mid dose group. The positive level of index 1.4 for cell counts has been exceeded in this dose group. Cell count index (test item concentration): 1.00 (0%) / 1.20 (10%) / 1.58 (30%) / 1.41 (100%).

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: modified LLNA; measurement of cell counts instead of radioactive labeling

Any other information on results incl. tables

The positive level of ear swelling has been exceeded in the mid dose group. This change is of statistical significance:

 

Ear swelling day 1 = 17.25 (0%) / 17.25 (10%) / 17.58 (30%) / 17.00 (100%); day 4 = 17.50 (0%) / 17.75 (10%) / 21.00 (30%) / 17.58 (100%); Index day 4 = 1.00 (0%) / 1.01 (10%) / 1.20 (30%) / 1.00 (100%)

A significant increase compared to vehicle treated animals regarding ear weights was also detected in the mid dose group: ear weight day 4 = 11.43 (0%) / 12.19 (10%) / 13.51 (30%) / 11.40 (100%); Index day 4 = 1.00 (0%) / 1.07 (10%) / 1.18 (30%) / 1.00 (100%).

The weight of the draining lymph nodes was not increased significantly: weight index 1.00 (0%), 1.07 (10%), 1.18 (30%), 1.18 (100%).

The body weights of the animals were not affected by any treatment. In the animals of the high dose group clinical findings (apathy, rough coat, poor general condition) were observed from day 2 until autopsy.

The EC1.4 value calculated is 20.53% for Mecoprop-P n-octyl ester.

Applicant's summary and conclusion

Executive summary:

A modified LLNA (IMDS; OECD TG 429) was performed on 6 female NMRI mice per dose group using epicutaneously applied test substance of 0% (vehicle control), 10%, 30% and 100%.

The study does point to a non-specific (irritant) and to a specific immuno-stimulating (sensitizing) potential of Mecoprop-P n-octyl ester. This applies to NMRI mice, for cell counts of the draining lymph nodes as well as ear swelling and ear weight indices evaluated after application of the formulated test item. The EC1.4 value calculated is 20.53% for Mecoprop-P n-octyl ester. The concentration of 10% turned out to be the NOEL for the parameters investigated in this study with respect to skin sensitization.