Reaction mass of 2-{[2-(2-hydroxyethoxy)ethoxy]carbonyl}benzoic acid and 2,2'-oxydiethanol

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:

guideline study

Remarks:

This study was condusted in compliance with the principles of GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

- modification: in addition, measurements of ear swelling and ear weight were done to discriminate the irritating potential from the sensitizing potential of the test substance (Integrated Model for the Differentiation of Skin reactions (IMDS))

Principles of method if other than guideline:

Modified LLNA (IMDS; Integrated Model for the Differentiation of Skin Reactions). Modifications are authorised 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:

2-(2-hydroxyethoxy)ethan-1-ol; 2-{[2-(2-hydroxyethoxy)ethoxy]carbonyl}benzoic acid

EC Number:

700-993-7

Molecular formula:

C12H14O6.C4H10O3

IUPAC Name:

2-(2-hydroxyethoxy)ethan-1-ol; 2-{[2-(2-hydroxyethoxy)ethoxy]carbonyl}benzoic acid

Test material form:

liquid

In vivo test system

Test animals

Species:

mouse

Strain:

NMRI

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Strain: Crl:NMRI BR
- Age at study initiation: 8 weeks
- Weight at study initiation: 26 - 32 g
- Housing: individual
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 6 days

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

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

0, 25 %, 50 % and 100 %

No. of animals per dose:

6

Details on study design:

TREATMENT PREPARATION AND ADMINISTRATION:
The test item in the formulation, the positive control or the vehicle were 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 25µl/ear. The used concentrations were based on the experiences with the test system and the toxic properties of the test substance.
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 draining lymph nodes (given as weight index compared to vehicle controls)
- cell counts in draining lymph nodes (given as cell count index compared to vehicle controls)
Stimulation indices were calculated by dividing the absolute weight or number of cell counts of the substance treated lymph nodes by the vehicle treated ones.
- ear swelling (given in 0.01 mm and as index)
- ear weight (given in mg/8 mm diameter punch and as index)
- 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 heterogenous (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 differentes 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 showed a clear sensitizing potential in the local lymph node assay (IMDS).

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks:

weight of draining lymph nodes

Value:

1

Test group / Remarks:

Vehicle control group, 6 animals

Results 2

Parameter:

SI

Remarks:

cell count in draining lymph node

Value:

1

Test group / Remarks:

Vehicle control group, 6 animals

Results 3

Parameter:

SI

Remarks:

Ear swelling

Value:

1

Test group / Remarks:

Vehicle control group, 6 animals

Results 4

Parameter:

SI

Remarks:

weight of draining lymph nodes

Value:

1.08

Test group / Remarks:

Dose 25% Ester PSA + DEG, 6 animals

Results 5

Parameter:

SI

Remarks:

cell count in draining lymph nodes

Value:

0.79

Test group / Remarks:

Dose 25% Ester PSA + DEG, 6 animals

Results 6

Parameter:

SI

Remarks:

Ear swelling

Value:

1

Test group / Remarks:

Dose 25% Ester PSA + DEG, 6 animals

Results 7

Parameter:

SI

Remarks:

weight of draining lymph nodes

Value:

1.32

Test group / Remarks:

Dose 50% Ester PSA + DEG, 6 animals

Results 8

Parameter:

SI

Remarks:

cell count in draining lymph nodes

Value:

1.24

Test group / Remarks:

Dose 50% Ester PSA + DEG, 6 animals

Results 9

Parameter:

SI

Remarks:

Ear swelling

Value:

0.99

Test group / Remarks:

Dose 50% Ester PSA + DEG, 6 animals

Results 10

Parameter:

SI

Remarks:

weight of draining lymph nodes

Value:

1.32

Test group / Remarks:

Dose 100% Ester PSA + DEG, 6 animals

Results 11

Parameter:

SI

Remarks:

cell count in draining lymph nodes

Value:

1.14

Test group / Remarks:

Dose 100% Ester PSA + DEG, 6 animals

Results 12

Parameter:

SI

Remarks:

Ear swelling

Value:

1

Test group / Remarks:

Dose 100% Ester PSA + DEG, 6 animals

Any other information on results incl. tables

Table 1: Summary of the LLNA/IMDS results with Ester PSA + DEG (means of 6 animals per group)

 

Parameter investigated	Vehicle control	Dose    25%	Dose 50 %	Dose 100 %	Positive control
Stimulation index: weight of draining lymph nodes	1.00	1.08	1.32	1.32	1.89 *
Stimulation index: cell count in draining lymph nodes	1.00	0.79	1.24	1.14	1.92 *
Ear swelling in 0.01 mm on day 4 (index)	17.83 (1.00)	17.92 (1.00)	17.67 (0.99)	17.75 (1.00)	23.67 * (1.33)
Ear weight in mg / 8 mm diameter punch on day 4 (index)	12.91 (1.00)	12.24 (0.95)	12.13 (0.94)	11.81# (0.91)	16.41 * (1.27)

* statistically significant increase (p <= 0.05)

# statistically significant decrease (p <= 0.05)

The body weights of the animals were not affected by any treatment.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Executive summary:

The test item was investigated in the modified local lymph node assay (LLNA-IMDS) on female mice according to OECD TG 429. Concentrations of 0 (vehicle control), 25 %, 50 % and 100 % formulated in dimethylformamide were tested. This study does neither point to a non-specific (irritant) nor to a specific immunostimulating (sensitizing) potential ofthe test item. These findings were verified by the results obtained with the positive control compound Alpha Hexyl Cinnamic Aldehyde.