Benzoic acid, 4-[4-(2-oxiranyl)butoxy]-, 4-methoxyphenyl ester

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 15, 2011 - July 04, 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

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:

-

EC Number:

435-360-1

EC Name:

-

Cas Number:

144447-11-0

Molecular formula:

C20H22O5

IUPAC Name:

4-methoxyphenyl 4-[4-(oxiran-2-yl)butoxy]benzoate

Details on test material:

- Name of test material (as cited in study report): Benzoic acid, 4-[4-(2-oxiranyl)butoxy]-, 4-methoxyphenyl ester
- Chemical name: 4-methoxyphenyl-4(5,6-epoxyhexyloxy)benzoate
- Physical state: White to off-white powder
- Storage condition of test material: At room temperature in the dark
- Stability under storage conditions: Stable
- pH: Not indicated
- Stability in dimethyl formamide: Not indicated
- Solubility in dimethyl formamide: Not indicated

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: Young adult animals (approx. 11 weeks old)
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean.
- Housing: Animals were group housed in labeled Makrolon cages (MIII type; height 18 cm) containing sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) was supplied as cage-enrichment. The acclimatization period was at least 5 days before the start of treatment under laboratory conditions. On Day 6, the animals were group housed in Makrolon MII type cages with a sheet of paper instead of sawdust and cage enrichment.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: The acclimatization period was at least 5 days before the start of treatment under laboratory conditions.
- Health inspection: A health inspection was performed prior to treatment, to ensure that the animals are in a good state of health. Special attention was paid to the ears, which were intact and free from any abnormality.

Results of analysis for diet (nutrients and contaminants), sawdust, paper and water were assessed and did not reveal any findings that were considered to have affected the study integrity. All certificates and results of analysis are retained in the NOTOX archives.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.3 – 22.0
- Humidity (%): 40 - 70% (actual range (REES): 42-68%)
List of protocol deviations:
1. From two days preceding start of the main study onwards, temperature and relative humidity were not recorded by the REES Centron Environmental Monitoring system.
Evaluation: Temperature and relative humidity data from an alternative (non-GLP) environmental recording system suggested that the temperature was within protocol specifications during that respective period, but relative humidity was outside these specifications (see separate deviation). A default set-up was used for maintaining environmental conditions during this period, and there were no indications among the animals which indicated that significant deviations from this set-up had occurred. Immediately before and after this period, temperature and relative humidity data registered by the Rees monitoring system were within protocol ranges. Overall, it was considered that during this period no variations in temperature and relative humidity occurred that would have adversely affected the study integrity based on laboratory historical data.
2. Temporary deviations from the maximum level of relative humidity occurred in the animal room.
Evaluation: Laboratory historical data do not indicate an effect of the deviations.

The study integrity was not adversely affected by the deviations.

- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: June 15, 2011 to July 04, 2011

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

0, 10, 25 and 50%

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
A pre-screen test was conducted in order to select the highest test substance concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give moderate irritation at the most (maximum grade 2) and/or an increase in ear thickness < 25%) and is the highest possible concentration that can technically be applied.

Two test substance concentrations were tested; a 50% and 25% concentration. The highest concentration was the maximum concentration as required in the test guidelines (undiluted for liquids, 50% for solids).

The test system, procedures and techniques were identical to those used in the main study except that assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected (in the range of 8 to14 weeks old). Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 12.5 cm). Ear thickness measurements were conducted using a digital thickness gauge prior to dosing on Days 1 and 3, and on Day 6. Animals were sacrificed after the final observation.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group.
If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer.
The results were evaluated according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2007) and the Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures.
The EC3 value (the estimated test substance concentration that will give a SI =3) was determined, using linear interpolation

ANIMAL ASSIGNMENT
Three groups of five animals were treated with one test substance concentration per group. One group of five animals was treated with vehicle.

TREATMENT PREPARATION AND ADMINISTRATION:
Test substance preparation: The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. No adjustment was made for specific gravity of the vehicle. Homogeneity was obtained to visually acceptable levels.
Rationale for vehicle: The vehicle was selected based on trial formulations performed at NOTOX and on test substance data supplied by the sponsor.

Induction - Days 1, 2 and 3:
The dorsal surface of both ears was epidermally treated (25 μL/ear) with the test substance concentration, at approximately the same time per day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing. The control animals were treated the same as the experimental animals, except that the vehicle was administered instead of the test substance.

Excision of the nodes - Day 6:
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 μCi of 3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US). After approximately five hours, all animals were killed by intraperitoneal injection with Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 mL PBS.

Tissue processing for radioactivity - Day 6:
A single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter 125 μm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) stored in the refrigerator until the next day.

Radioactivity measurements - Day 7
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactive measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

Observations:
Mortality/Viability: Twice daily.
Body weights: On Day 1 (pre-dose) and Day 6 (prior to necropsy).
Clinical signs: Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
Irritation: Once daily on Days 1-6 (on Days 1 - 3 immediately after dosing) according to the following numerical scoring system. Furthermore, a description of all other (local) effects was recorded.

Grading Irritation Reactions:
Erythema and eschar formation:
0: No erythema
1: Very slight erythema (barely perceptible)
2: Well-defined erythema
3: Moderate to severe erythema (beet redness) to slight eschar formation (injuries in depth)
4: Severe erythema (beet redness) to eschar formation preventing grading of erythema

Necropsy: All animals were subjected to necropsy for gross macroscopic examination.

Positive control substance(s):

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

Statistics:

Not performed.

Results and discussion

Positive control results:

The six-month reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity. See attached document 'Reliability check'.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: The SI values calculated for the substance concentrations 10, 25 and 50% were 3.4, 3.2 and 2.4 respectively.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 1032, 962 and 735 DPM respectively. The mean DPM/animal value for the vehicle control group was 302 DPM.

Any other information on results incl. tables

Tables and figures of the Pre-screen test and Main study have been included in the attached document "LLNA tables and figures".

Results Pre-screen test:

No irritation and no signs of systemic toxicity were observed in any of the animals examined. White test substance remnants were present on the ears of one or both animals at 25 and 50% between Days 1 and 3, which did not hamper the scoring of any skin reactions. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. Based on these results, the highest test substance concentration selected for the main study was a 50% concentration.

Other results - main study:

No irritation of the ears was observed in any of the animals examined.White test substance remnants were present on the ears of all animals at 25% (Days 1-3) and 50% (Days 1-4), which did not hamper the scoring of any skin reactions.

 

All auricular lymph nodes at 10 and 25% and most auricular lymph nodes at 50% appeared larger in size when compared to the other treated groups. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

 

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The slight body weight loss (1-2 g), noted in some experimental animals and a control animal, was considered not related to treatment with the test substance.

    

No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Remarks:

Migrated information

Conclusions:

Assessment of Contact Hypersensitivity to Benzoic acid, 4-[4-(2-oxiranyl)butoxy]-, 4-methoxyphenyl ester in the Mouse (Local Lymph Node Assay, 5 females/dose) was conducted according to OECD 429 guidelines and GLP principles.

The results show that the test substance elicits an SI ≥ 3. The EC3 value (the estimated test substance concentration that will give a SI =3) was established to be between 0 and 10%.

Based on these results, according to the recommendations made in the test guidelines, Benzoic acid, 4-[4-(2-oxiranyl)butoxy]-, 4-methoxyphenyl ester would be regarded as skin sensitizer.