Post crystallisation of ammonium sulfate aqueous phase products resulting from ammoniac neutralisation of sulfuric acid waste waters formed during methylmethacrylate synthesis

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 13 August 2009 to 15 January 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Compliant to GLP and testing guideline; adequate coherence between data, comments and conclusions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

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

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:

Post crystallization of ammonium sulphate aqueous phase products resulting from ammoniac neutralisation of sulphuric acid waste waters formed during methylmetacrylate synthesis

IUPAC Name:

Post crystallization of ammonium sulphate aqueous phase products resulting from ammoniac neutralisation of sulphuric acid waste waters formed during methylmetacrylate synthesis

Details on test material:

- Physical state: brown black liquid
- Lot/batch No.: prélévement SR320 du 1 juillet 2009
- Expiration date of the lot/batch: 01/07/2011
- Storage condition of test material: at room temperature
- Purity/Impurities: not applicable (complex composition)

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Breeder: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approximately 11 weeks old
- Weight at study initiation: 23.7 +- 1.0 g
- Housing: housed individually in disposal crystal polystyrene cages (22 cm x 8.5 cm x 8 cm). Each cage contained autoclaved sawdust.
- Diet (e.g. ad libitum): free access to SSNIFF R/M-H pelleted maintenance diet
- Water (e.g. ad libitum): free access to tap water (filtered using a 0.22 micron filter)
- Acclimation period: at least 5 days before the beginning of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 2 °C
- Humidity (%): 30 to 70%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12h/12h (7:00 - 19:00)

IN-LIFE DATES: From: 14 August 2009 To: 31 August 2009

Study design: in vivo (LLNA)

Vehicle:

other: (Ethanol/purified water 50/50 w/w)

Concentration:

0.5, 1, 2.5, 5 and 10%

No. of animals per dose:

4 animals per dose

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: not soluble in AOO at 25%. Heterogeneous emulsion at 50% and 25% in DMF, MEK, PG, DMSO and 1% Pluronic L92. Heterogeneous suspension at 50% and 25% in Ethanol/purified water (50/50, w/w). Solution at 10% in Ethanol/purified water (50/50, w/w).

- Irritation: not excessively irritant, whatever the concentration
- Lymph node proliferation response: incorporation of tritiated methyl thymidine

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: SI >= 3 with exclusion of excessive irritation

TREATMENT PREPARATION AND ADMINISTRATION:
On days 1, 2 and 3, a dose-volume of 25 µL of the control or dosage form preparations was applied to the dorsal surface of both ears, using an adjustable pipette fitted with a plastic tip.

Positive control substance(s):

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

Results and discussion

Positive control results:

HCA at the concentration of 25%: a moderate increase in cellularity and a stimulation index exceeding the threshold value of 3 (SI = 14.34) were noted. The study was therefore considered valid.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: Group 2: test item 0.5%: 0.75 Group 3: test item 1%: 1.01 Group 4: test item 2.5%: 0.91 Group 5: test item 5 %: 0.83 Group 6: test item 10%: 1.03

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Group 1: Vehicle: 474.60 Group 2: test item 0.5%: 355.10 Group 3: test item 1%: 480.36 Group 4: test item 2.5%: 433.73 Group 5: test item 5%: 391.62 Group 6: test item 10%: 490.62

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

The test item did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay.

Executive summary:

The potential of the test item to induce delayed contact hypersensitivity using the murine Local Lymph Node Assay (LLNA) was evaluated.Evaluation of local irritation was also carried out in parallel. This study was conducted in compliance with the principles of Good Laboratory Practice Regulations.

A preliminary test was first performed in order to define the concentrations of test item to be used in the main test.

In the main test, twenty-eight female CBA/J mice were allocated to seven groups:

× five treated groups of four animals receiving the test item at the concentration of 0.5, 1, 2.5, 5 or 10% in a mixture Ethanol/purified water (50/50, w/w),

× one negative control group of four animals receiving the vehicle (mixture Ethanol/purified water (50/50, w/w)),

× one positive control group of four animals receiving the reference item, a-hexylcinnamaldehyde (HCA), a moderate sensitizer, at the concentration of 25% in a mixture acetone/olive oil (4/1,v/v).

During the induction phase, the test item, vehicle or reference item was applied over the ears (25 µL per ear) for 3 consecutive days (days 1, 2 and 3). After 2 days of resting, the proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of tritiated methyl thymidine (day 6). The obtained values were used to calculate stimulation indices (SI).

The irritant potential of the test item was assessed in parallel by measurement of ear thickness on days 1, 2, 3 and 6.

The test item was soluble in Ethanol/purified water (50/50, w/w) at the maximum concentration of 10%.

Consequently, the concentrations selected for the preliminary test were 1, 2.5, 5 and 10%.

Since the test item was not excessively irritant in the preliminary test, the highest concentration retained for the main test was the maximal practicable concentration (10%). No mortality or clinical signs were observed during the study. No cutaneous reactions or notable increase in ear thickness were observed in the animals of the treated groups. A significant lymphoproliferation was noted in the positive control group given HCA. The study was therefore considered valid. No notable lymphoproliferation was noted at any of the tested concentrations (SI from 0.75 to 1.03).

Under the experimental conditions of this study, the test item did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay.