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Toxicological information

Skin sensitisation

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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: Study conducted according to GLP and OECD guideline 429.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report date:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Ammonium carbamate
EC Number:
214-185-2
EC Name:
Ammonium carbamate
Cas Number:
1111-78-0
Molecular formula:
CH3NO2.H3N
IUPAC Name:
ammonium carbamate
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Purity/Composition:
>99% ammonium carbamate (reported as 43.6 g/100g NH3 and 56.3 g/100g CO2
0.1 g/100g H2O
0.1 mg/kg Fe
32 mg/kg Sulphated ash

Storage:
At room temperature in the dark

pH: 10

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J, inbred, SPF
Sex:
female
Details on test animals and environmental conditions:
Source:
Janvier, Le Genest-Saint-Isle, France

Age and body weight:
Young adult animals (approx. 10 weeks old) were selected. Body weight variation was within +/- 20% of the sex mean.

Identification Tail:
mark with marker pen.

Health inspection:
Upon receipt of the animals and prior to dosing. Prior to dosing, it was ensured that the animals were without any observable skin lesions and the ears are intact and free from any abnormality.

Conditions:
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

Accommodation:
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) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd, USA) were 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:
Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).

Water:
Free access to tap water.

Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures.
There were no findings that could interfere with the study.

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
10, 25, 50%
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TEST:
Conducted in order to select the highest test substance concentration to be used in the main study. The test system, procedures and techniques were identical to those used in the main study. Two young adult animals per concentration were selected (in the range of 8 to14 weeks old). Three test substance concentrations (10, 25 and 50%) were tested. The highest concentration was the maximum concentration as required in the test guidelines (undiluted for liquids, 50% for solids). One group of two animals was treated with the positive control substance (0.5% 1-Chloro-2,4-dinitrobenzene (DNCB)). Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 14 cm). Ear thickness measurements were conducted using a digital thickness gauge prior to dosing on Days 1 and 3, and on Day 6. For all animals, the draining auricular lymph nodes were excised on Day 6, and processed for radioactivity measurements as specified for the Main study.

MAIN STUDY:
Three groups of five animals were treated with one test substance concentration per group. The highest test substance concentration was selected from the pre-screen test. One group of five animals was treated with vehicle, and another group of five animals was treated with the positive control substance.

- Induction (Days 1,2 and 3): Dorsal surface of both ears topically treated (25 μL/ear) with the test substance concentration, at approximately the same time on each day. The vehicle and positive control animals were treated in the same way as the experimental animals, except that the vehicle and/or positive control substance 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 (0.2 mL/animal) 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) and stored in the refrigerator (prescreen-test) or at room temperature (main study) 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 (2x daily), body weights (on day 1 before dosing and day 6 prior to necropsy), irritation (1x daily on days 1-6).
- Interpretation: 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. Consideration was given to the EC3 value (the estimated test substance concentration that will give a SI =3)
Positive control substance(s):
other: 1-Chloro-2,4-Dinitrobenzene (DNCB)

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: - 10%: 1.1 - 25% 1.2 - 50%: 0.6
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/animal values: - 10%: 386 DPM - 25%: 411 DPM - 50%: 228 DPM

Any other information on results incl. tables

No irritation of the ears was observed in any of the animals examined. No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The body weight loss noted for some animals across the dose groups was considered not toxicologically significant since the changes were slight in nature and no concentration-related incidence was apparent.

All auricular lymph nodes of the animals treated at 10, 25 or 50% and of control group animals were considered normal in size. The auricular lymph nodes of the positive control group animals appeared larger in size as compared to the other treated groups. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Based on these results, Ammonium Carbamate is not regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test substance does not have to be classified for sensitization by skin contact according to Regulation (EC) No 1272/2008 (CLP).
Executive summary:

Ammonium carbamate was tested for its potential to cause skin sensitization in a GLP-compliant local lymph node assay performed according to OECD guideline 429. In the main study, 3 experimental groups of 5 female CBA/J mice were treated with test substance concentrations of 10, 25 and 50% on 3 consecutive days by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (propylene glycol) and another group of five animals received a positive control substance (DNCB).

Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal.

After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of Disintegrations Per Minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

No irritation of the ears was observed in any of the animals examined.

All auricular lymph nodes of the animals treated at 10, 25 or 50% and of control group animals were considered normal in size. The auricular lymph nodes of the positive control group animals appeared larger in size as compared to the other treated groups. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 386, 411 and 228 DPM respectively. The mean DPM/animal value for the vehicle control group was 353 DPM and a mean DPM/animal value of 16082 DPM was obtained for the positive control group.

The SI values calculated for the substance concentrations 10, 25 and 50% were 1.1, 1.2 and 0.6 respectively.

Since there was no indication that the test substance elicits an SI ≥ 3 when tested up to 50%, Ammonium Carbamate was considered not to be a skin sensitizer.