Ammonium carbonate

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

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: Test method in accordance with OECD 429 and EU Method B.42. GLP study.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: ELEVAGE JANVIER, Route des Chènes Secs B.P. 4105. 53940 LE GENEST-ST-ISLE, France
- Age at study initiation: 9 weeks old
- Weight at study initiation: 20.0-21.6 grams
- Housing: Group caging. Cage type: Type II. polypropylene / polycarbonate with bedding of certified wood chips
- Diet (e.g. ad libitum): Ad libitum.
- Water (e.g. ad libitum): Ad libitum.
- Acclimation period: 13 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 15-20 air exchange/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Vehicle:

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

Concentration:

50, 25 and 10 % w/v test item.

No. of animals per dose:

4 animals per group

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: Based on the observation of the solubility test, the maximum concentration of test item in an acceptable solvent was established at 50 (w/v) % concentration using AOO (acetone:olive oil 4:1 (v:v) mixture).
- Irritation: A Preliminary Irritation/Toxicity Test was performed using 2 mice per dose, at test item concentrations of 50 and 25 (w/v) % in AOO. The preliminary experiment was conducted in a similar experimental manner to the main study, but it was terminated on Day 6. Mice were observed daily for any clinical signs of systemic toxicity or local irritation (scoring according to OECD Guideline 404) and at the application site. Ear thickness was measured using a thickness gauge on Day 1, 3 and 6. No effects were observed, and therefore 50 and 25 (w/v) % doses were considered to be acceptable for the main test.
- Lymph node proliferation response: The radioactive proliferation assay was not performed.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Proliferation assay.
- Criteria used to consider a positive response: The test item is regarded as a sensitizer if both of the following criteria are fulfilled:
That exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
The data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
Animals were topically dosed with 25 µL of the appropriate formulation (0, 50, 25 and 10 % w/v) using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.
During the study (Day 1 to Day 6) each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Individual body weights were recorded on Days 1 and 6.
On Day 6, each mouse was intravenously injected via the tail vein with 250 µL of sterile PBS (phosphate buffered saline) containing approximately 20 µCi of 3HTdR. Five hours after mice were euthanized. Then, the draining auricular lymph nodes were excised and the nodes removed. Cell suspension (SCS) of pooled lymph node cells (LNCs) were prepared. After overnight (approximately 18 hours) incubation at 2-8 ºC, the vials were loaded into a β-scintillation counter and 3HTdR incorporation was measured as the number of radioactive disintegrations per minute (DPM) (background level was also measured).

Positive control substance(s):

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

Positive control results:

No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A significant lymphoproliferative response (stimulation index value of 10.7) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay. Furthermore, the DPN values were within the historical control range.

Parameter:

SI

Remarks on result:

other: Negative (vehicle) control (AOO): 1.0 Ammonium carbonate e 50 (w/v) % in AOO: 1.3 Ammonium carbonate 25 (w/v) % in AOO: 1.7 Ammonium carbonate 10 (w/v) % in AOO: 1.6 Positive control (25 (w/v) % HCA in AOO): 10.7

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Negative (vehicle) control (AOO): 1131.5 Ammonium carbonate 50 (w/v) % in AOO: 1516.5 Ammonium carbonate 25 (w/v) % in AOO: 1922.5 Ammonium carbonate 10 (w/v) % in AOO: 1787.5 Positive control (25 (w/v) % HCA in AOO): 12132.5

No mortality or signs of systemic toxicity were observed during the study. There were no indications of any irritancy at the site of application. No treatment related effects were observed on animal body weights.

DPM, DPN and Stimulation Index Values for all Groups:

Test Group Name	Measured DPM / group	DPM	Number of lymph nodes	DPN	Stimulation Index
Background	36.5	-	-	-	-
(5 (w/v) % TCA)
Negative (vehicle) control	1168	1131.5	8	141.4	1.0
(AOO)
Ammonium carbonate	1553	1516.5	8	189.6	1.3
50 (w/v) % in AOO
Ammonium carbonate	1959	1922.5	8	240.3	1.7
25 (w/v) % in AOO
Ammonium carbonate	1824	1787.5	8	223.4	1.6
10 (w/v) % in AOO
Positive control	12169	12132.5	8	1516.6	10.7
(25 (w/v) % HCA in AOO)

All validity criteria were fulfilled.

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Ammonium carbonate was shown to have no sensitization potential (non-sensitizer) in the Local Lymph Node Assay. The observed stimulation index values were 1.3, 1.7 and 1.6 at concentrations of 50, 25 and 10 (w/v) %, respectively.

Executive summary:

A skin sensitization Local Lymph Node Assay was performed on Ammonium carbonate in accordance with OECD Guideline 429 and EU method B.42. Based on the preliminary compatibility test the highest achievable concentrations was 50 (w/v) % in AOO (acetone: olive oil 4:1 (v:v) mixture) as vehicle. No effects were observed in the preliminary irritation/toxicity test at 50 and 25% (w/v) % in AOO. In the main assay, 4 female mice per dose were dermally exposed to 25 µL/ear application of 0 (vehicle control), 50, 25, 10 (w/v) % Ammonium carbonate , 25 (w/v) % HCA (positive control) for 3 consecutive days. There was no treatment for another 3 consecutive days. On Day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI). No mortality or systemic clinical signs were observed during the study. No treatment related effects were observed on animal body weights in any treated groups. There were no indications of any irritancy at the site of application. The observed stimulation index values were 1.3, 1.7 and 1.6 at concentrations of 50, 25 and 10 (w/v) %, respectively. All the validity criteria were fulfilled. In conclusion, under the conditions of the present assay Ammonium carbonate , tested in a suitable vehicle, was shown to have no sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Migrated from Short description of key information:
Ammonium carbonate was shown to have no sensitization potential (non-sensitizer) in the Local Lymph Node Assay. The observed stimulation index values were 1.3, 1.7 and 1.6 at concentrations of 50, 25 and 10 (w/v) %, respectively.

Justification for classification or non-classification

The substance is not classified.