Urea, polymer with ethanedial

Endpoint summary

Description of key information

Skin sensitization in vivo: Key study. OECD 429 Guideline. GLP study.The EC3 value is 28.6%. The test item is classified as skin sensitizer.

Skin sensitisation in vitro: Data waiving: Study scientifically not necessary / other information available: In accordance with the Annex VII column 2 of REACH (amended on 20 September 2016), an in vitro or in chemico skin sensitisation study does not need to be conducted because adequate data from in vivo skin sensitisation study are available.

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:

From 25 May 2016 to 31 May 2016

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)

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/Ca

Remarks:

CBA/CaOlaHsd mice

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS- Source: Envigo, San Pietro al Natisone (UD), Zona Industriale Azzida, 57, 33049 Italy- Females (if applicable) nulliparous and non-pregnant: yes- Age at study initiation: 12 weeks old - Weight at study initiation: 19.6 – 21.6 grams. The weight variation in animals in the study did not exceed± 20 % of the mean weight.- Housing: Group caging / mice were provided with glass tunnel-tubes. Type II. polypropylene / polycarbonate cages- Diet (e.g. ad libitum): ad libitum. Sniff® SM Rat/Mouse – “Breeding & Maintenance, 15 mm, autoclavable Complete diet for rats/mice” (Batch number: 540 5117, Expiry date: 31 July 2016 and Batch number: 278 5652 Expiry date: 30 November 2016)- Water (e.g. ad libitum): ad libitum. Municipal supply from 500 mL bottle.- Acclimation period: 35 days- Indication of any skin lesions: Only healthy animals were used for the study. Health status was certified by the veterinarian.ENVIRONMENTAL CONDITIONS- Temperature (°C): 19.8 – 25.9°C- Humidity (%): 29- 80 %- Air changes (per hr): 15-20 air exchanges/hour- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.- IN-LIFE DATES: From: 20 April 2016 To: 30 May 2016

Vehicle:

other: 1% Pluronic

Concentration:

25, 50 and 100 % w/v in vehicle

No. of animals per dose:

4 animals per dose

Details on study design:

PRE-SCREEN TESTS:- Compound solubility: The solubility of the test item was examined. The following standard OECD vehicles were assessed: AOO (acetone:olive oil 4:1 (v:v) mixture), N,N-dimethylformamide, Methyl ethyl ketone, Propylene glycol, Dimethyl sulfoxide and 1% aqueous Pluronic® PE9200. The best vehicle was considered to be 1% Pluronic. The highest achievable concentration was 100% (w/v).- Irritation: It was evaluated on CBA/CaOlaHsd mice using two doses (2 animals/dose) at test item concentrations of 100% (w/v) and 50% (w/v) in 1% Pluronic. The preliminary experiment was conducted in a similar experimental manner to the main study, but it was terminated on Day 6 and the radioactive proliferation assay was not performed. - Systemic toxicity: No mortality was observed. Test item precipitate or minimal amount of test item precipitate was observed on the ears of the animals in both dose groups on Days 1-3. Alopecia around the ears was observed for both animals of the 100% (w/v) dose group on Day 2 and for both animals of the 50% (w/v) dose group on Days 4-6. Extensive alopecia was observed for both animals of the 100% (w/v) dose group on Days 3-6. Slightly rigid ear was observed for both animals of the 100% (w/v) dose group on Day 2. No marked body weight loss (>5%) was detected in the experimental animals.- Draining auricular lymph nodes: they were larger than normal in the 100% (w/v) dose group and they were normal in the 50% (w/v) dose group (subjective judgement by analogy with observations of former experiments).- Ear thickness measurements: The ear thickness values and ear punch weights were within the acceptable range.- Erythema scores: The erythema score was 0 in all cases.MAIN STUDYBased on the results of pre-screen tests, 100% (w/v) dose group is selected as top dose for the main test. 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).Animal assignement and treatment- Criteria used to consider a positive response: The test item is regarded as a sensitizer if both of the following criteria are fulfilled:a. 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.b. 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 administrationIn the main study, three doses of the test item and one negative and one positive control were tested. Animals were topically dosed with 25 μL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutivedays (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.Proliferation assay:On Day 6, ach mouse was intravenously injected via the tail vein with 250 μL of sterile PBS containing approximately 20 μCi of 3HTdR. Onceinjected, the mice were left for 5 hours (± 30 minutes). After this period mice were euthanized by asphyxiation with ascending doses of carbon dioxide. The draining auricular lymph nodes were removed and single cells suspensions of pooled lymph node cells were prepared and after overnight (approximately 18 hours) incubation at 2-8 °C, the 3HTdR incorporation was measured (10-minute measurement). Background level was also measured in duplicates.Observations: Clinical Observations and measurement of body weightDuring the study (Day 1 to Day 6) each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Individual records were maintained.Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g.

Positive control substance(s):

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

Positive control results:

The result of the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. The positive control substance was examined at a concentration of 25 % in the relevant vehicle (1% Pluronic) using CBA/CaOlaHsd mice. No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A lymphoproliferative response in line with historic positive control data (stimulation index value of 6.7) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay. Furthermore, the DPN values observed for the positive control substance in this experiment were within the historical control range.

Key result

Parameter:

EC3

Remarks:

(%)

Value:

ca. 28.6

Parameter:

SI

Value:

ca. 2.3

Test group / Remarks:

25 (w/v) % in 1% Pluronic

Parameter:

SI

Value:

ca. 7.2

Test group / Remarks:

50 (w/v) % in 1% Pluronic

Parameter:

SI

Value:

ca. 14.2

Test group / Remarks:

100 % (undiluted)

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA: Test item 100% (w/v) : 35513 DPMTest item 50% (w/v) in 1% Pluronic: 17983 DPMTest item 25% (w/v) in 1% Pluronic: 5890 DPMDETAILS ON STIMULATION INDEX CALCULATION:The stimulations index values were 14.2, 7.2 and 2.3 at a concentration of 100, 50 and 25% (w/v), respectively. The calculated EC3 value was calculated to be 28.6% (w/v)CLINICAL OBSERVATIONS: No mortality or systemic clinical signs were observed during the study. Extensive alopecia around the ears was observed at 100% (w/v) concentration on Days 2-6 and alopecia around the ears was observed for three animals of the 50% (w/v) dose group on Days 4-6. Minimal amount of test item precipitate was observed on the ears of the animals at 100% (w/v) concentration on Days 1-3. BODY WEIGHTS: No treatment related effects were observed on the mean body weight of the groups, however, marked body weight loss (>5%) was detected for one animal of the negative control group and for one animal of the 50% (w/v) dose group. These changes are considered as animal variability.OTHER: The appearance of the lymph nodes was slightly enlarged than normal in the 50% (w/v) dose group and they were larger than normal in the 100% (w/v) dose group and in the positive control group. Normal lymph nodes were observed in the 25% (w/v) dose group and in the negative control group.

Main test: DPM, DPN and Stimulation Index Values for all Groups

Test Group Name	Measured DPM / group	DPM	Number of lymph nodes	DPN	Stimulation Index
Background (5 % (w/v) TCA)	35 36	-	-	-	-
Negative control (1% Pluronic)	2531	2495.5	8	311.9	1.0
Nopcote 1661 100 % (undiluted)	35513	35477.5	8	4434.7	14.2
Nopcote 1661 50 (w/v) % in 1% Pluronic	17983	17947.5	8	2243.4	7.2
Nopcote 1661 25 (w/v) % in 1% Pluronic	5890	5854.5	8	731.8	2.3
Positive control (25 % (w/v) HCA in 1% Pluronic)	16716	16680.5	8	2085.1	6.7

Notes:

1.         DPM (Disintegrations Per Minute)

2.         DPN (Disintegrations Per Node) = DPM divided by the number of lymph nodes.

3.         Stimulation Index = DPN of a treated group divided by DPN of the appropriate control group.

Test item Stimulation Index Values: please see attached Graph.

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

Under these test conditions, an EC3 value of 28.6% was calculated. Therefore, the test item is classified as skin sensitizer.

Executive summary:

The skin sensitisation potential of the test item was evaluated in accordance with the OECD 429 Guideline with GLP. The test item was supplied as a 45.4% water solution; it was freeze-dried to obtain the 100% concentration. A Preliminary Irritation/Toxicity Test was performed in CBA/CaOlaHsd mice using two doses (2 animals/dose): 100% (w/v) and 50% (w/v) in 1% Pluronic. Based on its results, the 100% (w/v) was selected as top dose for the main test. In the main assay, 20 female CBA/CaOlaHsd mice were used (n=4/group): three groups test item at 100%, 50% and 25% (in 1% Pluronic), a negative control group (vehicle) and a positive control group (25 % HCA in 1% Pluronic). The test item was applied on the dorsal surface of ears of experimental animals on Days 1, 2 and 3. There was no treatment on Days 4, 5 and 6. The cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the stimulation indices (SI) were calculated. No mortality or systemic clinical signs were observed during the study. Extensive alopecia around the ears was observed at 100% (w/v) concentration on Days 2-6 and alopecia around the ears was observed for three animals of the 50% (w/v) dose group on Days 4-6. The stimulation index values were 14.2, 7.2 and 2.3 at concentrations of 100% (w/v), 50% (w/v) and 25% (w/v), respectively. The calculated EC3 value is 28.6 %(w/v). Under these test conditions, the test item was shown to have sensitization potential. The test item is classified as skin sensitizer.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Skin sensitization: Key study. The study was performed according to OECD 429 Guideline with GLP. The test item was shown to have sensitization potential in the Local Lymph Node Assay. The calculated EC3 value is 28.6 %(w/v). The test item is classified as skin sensitizer.

Skin sensitisation in vitro: Data waiving: Study scientifically not necessary / other information available: In accordance with the Annex VII column 2 of REACH (amended on 20 september 2016), an in vitro or in chemico skin sensitisation study does not need to be conducted because adequate data from in vivo skin sensitisation study are available.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available data, the substance is classified for skin sensitizer Category 1 (sub-category 1B) according to CLP Regulation no. 1272/2008.