N-carbamoylmethyliminodi(acetic acid)

Endpoint summary

Description of key information

The observed stimulation index values in an OECD 429 study were 1.1, 0.6 and 1.0 at test item concentrations of 5 %, 2.5 % and 1 % (w/v), respectively. Therefore, the test item was considered to be not a skin sensitizer (reference 7.4.1 -1].

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

Study period:

2016-09-21 to 2016-09-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

22th July 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

20 July 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/Ca Ola Hsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: TOXI COOP ZRT. Cserkesz u. 90., 1103 Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals: SPF
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 16.9 - 20.3 g
- Fasting period before study: no
- Housing: group caging (4 mice/cage), Type II. Polypropylene / polycarbonate cages
- Diet: ad libitum, ssniff® Rat/Souris-Elevage E complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany
- Water: ad libitum, tap water
- Acclimation period: 21 days
- Indication of any skin lesions: Only healthy animals (and not showing any sign of skin lesion) were used.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3
- Humidity (%): 30 - 70
- Air changes (per hr): not available
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

other: 1N NaOH together with aqueous 1 % (w/v) Pluronic®PE 9200

Concentration:

1.0, 2.5, 5 %

No. of animals per dose:

4 main test, 2 in DRF

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: No or not adequate solubility was observed with standard vehicles at 2.5 % (w/v) concentration and above. Based on existing information, the maximum solubility in 1N NaOH was evaluated. The test item was completely soluble in 1N NaOH at a maximum concentration of 12.5 % (w/v). In the LLNA, control animals (used for calculation of test item SI values) should be treated with the relevant vehicle. Due to extreme pH (base) 1N NaOH alone cannot be applied on
the ears of animals hence formulations, prepared from the previously written formulation by dilution with Plu (not pH adjusted), was evaluated. The highest adequate concentration (using the recommended concentration series) resulting in acceptable pH and adequate solubility of the test item was 5 % (w/v).
- Irritation: No sign of significant irritation or any other local effect were observed at concentrations of 1.0, 2.5 and 5 % in a preliminary test.
- Systemic toxicity: No mortality, significant treatment related effect on the body weights or any other sign of systemic toxicity were observed.
- Ear thickness measurements: No changes in ear thickness were observed for the three concentration 1.0, 2.5 and 5 % in a preliminary test.
- Erythema scores: all 0 for all concentrations, mice and time points (days 1, 2, 3, 4, 5, 6)

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: animals were assigned randomly achieving body weight homogeneity between groups
- Criteria used to consider a positive response: stimulation index of one concentration > 3

TREATMENT PREPARATION AND ADMINISTRATION: Formulations (apparently solutions) were prepared as follows: 0.5 g test item was dissolved in 1N NaOH in a final volume of 4 mL using volumetric vials and mechanical agitation. This solution was supplemented to 10 mL with aqueous 1 % (w/v) Pluronic®PE 9200 (Plu; pH adjusted) to achieve 5 % (w/v) test concentration (stock solution). Further test concentrations were diluted from this stock solution with Plu. Formulations were freshly prepared just before the treatments. The pH value of each final formulation was recorded.
Each mouse was topically treated with 25 μL of the appropriate formulations of the test item, the positive control substance or the vehicles using a pipette, on the dorsal surface of each ear. After the treatments animals were returned to their cages. 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.

Positive control substance(s):

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

Statistics:

No statistical analysis was performed.

Positive control results:

No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group. Significant lymphoproliferative response (SI ≥ 3) was noted for HCA (SI = 9.3).

Parameter:

SI

Value:

1

Test group / Remarks:

1 %

Parameter:

SI

Value:

0.6

Test group / Remarks:

2.5 %

Key result

Parameter:

SI

Value:

1.1

Test group / Remarks:

5 %

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
Disintegration per minute (per mouse (average)):
vehicle control: 450.8
1.0 %: 430.8
2.5 %: 283.0
5.0 % 517.0

DETAILS ON STIMULATION INDEX CALCULATION: ratio of DPM/mouse of test concentrations and vehicle control

EC3 CALCULATION: not applicable, as no SI over 3

CLINICAL OBSERVATIONS: no irritation or other effects observed

BODY WEIGHTS: no treatment related effects

SIGNS OF TOXICITY: no confounding effects of irritation or systemic toxicity were observed during the main test

Interpretation of results:

GHS criteria not met

Conclusions:

The observed stimulation index values were 1.1, 0.6 and 1.0 at test item concentrations of 5 %, 2.5 % and 1 % (w/v), respectively. Therefore, the test item was considered to be not a skin sensitizer.

Executive summary:

A study according to OECD 429 was conducted to evaluate the skin sensitization potential of the test item following dermal exposure in the Local Lymph Node Assay. A formulation evaluation and a Dose Range Finding test (DRF) were performed to find an appropriate vehicle and the maximum applicable concentration according to the relevant guidelines. Solubility of the test item in vehicles preferred in the LLNA was evaluated. No adequate solubility was achieved with these vehicles, but the test item was completely soluble in 1N NaOH at a maximum concentration of 12.5 % (w/v). Due to extreme pH (base) 1N NaOH alone could not be applied on the ears of animals as vehicle control hence test item formulation was prepared by solution of the test item in 1N NaOH followed by dilution with aqueous 1 % (w/v) Pluronic®PE 9200 to achieve a concentration of 5 % (w/v). Lower test concentrations were diluted with aqueous 1 % (w/v) Pluronic®PE 9200. According to results of the DRF (where no adverse effect was observed up to this maximum concentration) the test item was examined in the main test as 5 %, 2.5 % or 1 % (w/v) formulations. Appropriate positive control (α-Hexylcinnamaldehyde, HCA) and its respective negative (vehicle) control (Acetone:Olive oil 4:1 (v/v) mixture, AOO) were also employed. Aqueous 1 % (w/v) Pluronic®PE 9200 adjusted with 1N NaOH to a pH similar to the test item formulations (Plu) was used as negative control for the test item. The positive control item (25 % (w/v) HCA in AOO) induced significant stimulation over the relevant control (SI = 9.3) thus confirming the validity of the assay. No mortality was observed during the main test. No significant, treatment related effect on body weights or any other sign of systemic toxicity were observed in any treatment group. No signs of irritation (monitored by erythema scoring) or any other local effect were observed at the treatment site (ears) in any treatment group. No significantly increased lymphoproliferation (indicated by an SI ≥ 3) compared to the relevant control (Plu) was noted for ADA at the applied test concentrations. The observed stimulation index values were 1.1, 0.6 and 1.0 at test item concentrations of 5 %, 2.5 % and 1 % (w/v), respectively. No significant dose-response relationship was observed. Accordingly, the test item was considered to be not a skin sensitizer. In conclusion, under the conditions of the present assay, the test item formulated in 1 N NaOH and aqueous 1 % (w/v) Pluronic®PE 9200 and tested at the maximum feasible concentration of 5 % (w/v, based on solubility) and also at concentrations of 2.5 % or 1 % (w/v) was shown to have no skin sensitization potential in the Local Lymph Node Assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

A study according to OECD 429 was conducted to evaluate the skin sensitization potential of the test item following dermal exposure in the Local Lymph Node Assay. A formulation evaluation and a Dose Range Finding test (DRF) were performed to find an appropriate vehicle and the maximum applicable concentration according to the relevant guidelines. Solubility of the test item in vehicles preferred in the LLNA was evaluated. No adequate solubility was achieved with these vehicles, but the test item was completely soluble in 1N NaOH at a maximum concentration of 12.5 % (w/v). Due to extreme pH (base) 1N NaOH alone could not be applied on the ears of animals as vehicle control hence test item formulation was prepared by solution of the test item in 1N NaOH followed by dilution with aqueous 1 % (w/v) Pluronic®PE 9200 to achieve a concentration of 5 % (w/v). Lower test concentrations were diluted with aqueous 1 % (w/v) Pluronic®PE 9200. According to results of the DRF (where no adverse effect was observed up to this maximum concentration) the test item was examined in the main test as 5 %, 2.5 % or 1 % (w/v) formulations. Appropriate positive control (α-Hexylcinnamaldehyde, HCA) and its respective negative (vehicle) control (Acetone:Olive oil 4:1 (v/v) mixture, AOO) were also employed. Aqueous 1 % (w/v) Pluronic®PE 9200 adjusted with 1N NaOH to a pH similar to the test item formulations (Plu) was used as negative control for the test item. The positive control item (25 % (w/v) HCA in AOO) induced significant stimulation over the relevant control (SI = 9.3) thus confirming the validity of the assay. No mortality was observed during the main test. No significant, treatment related effect on body weights or any other sign of systemic toxicity were observed in any treatment group. No signs of irritation (monitored by erythema scoring) or any other local effect were observed at the treatment site (ears) in any treatment group. No significantly increased lymphoproliferation (indicated by an SI ≥ 3) compared to the relevant control (Plu) was noted for ADA at the applied test concentrations. The observed stimulation index values were 1.1, 0.6 and 1.0 at test item concentrations of 5 %, 2.5 % and 1 % (w/v), respectively. No significant dose-response relationship was observed. Accordingly, the test item was considered to be not a skin sensitizer. In conclusion, under the conditions of the present assay, the test item formulated in 1 N NaOH and aqueous 1 % (w/v) Pluronic®PE 9200 and tested at the maximum feasible concentration of 5 % (w/v, based on solubility) and also at concentrations of 2.5 % or 1 % (w/v) was shown to have no skin sensitization potential in the Local Lymph Node Assay.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on skin sensitisation, the test item does not require classification according to Regulation (EC) No 1272/2008 (CLP), as amended for the fifteenth time in Regulation (EU) No 2020/1182.