Urea, reaction products with formaldehyde, glyoxal and methanol

Endpoint summary

Description of key information

The test item did not reveal skin sensitizing effects in the tested animals (mice).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

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)

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 000161MCA0

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River, Reserarch Models and Services, Sulzfeld, Germany
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 16.4 g - 20.2 g
- Housing:single housed
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 -70
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

other: 1% Pluronic L 92 Surfactant in highly de-ionized water

Concentration:

60 % w/w in 1% aqueous Pluronic

No. of animals per dose:

5

Details on study design:

Groups of 5 female CBA/J mice each were treated with a 60% w/w preparation of the test substance in 1% aqueous Pluronic® or with the vehicle alone. The 60% preparation was the maximum technically applicable concentration due to the sticky properties of the test substance.

The study was carried out as a limit test, using 1 test group and 1 control group. Each test animal was applied with 25 µL per ear of the testsubstance preparation to the dorsum of both ears for three consecutive days. The control group was treated with 25 µL per ear of the vehicle alone.

Three days after the last application the mice were injected intravenously with 20 µL of ³H-thymidine in 250 µL of sterile saline into a tail vein. About 5 hours after the ³H-thymidine injection, the mice were sacrificed and the auricular lymph nodes were removed. The weights of each animal’s pooled lymph nodes were determined. Thereafter lymph nodes were pooled group wise and further evaluated by measuring their cellular content and ³H-thymidine incorporation into the lymph node cells (indicators of cell proliferation). Moreover, a defined area with a diameter of 0.8 cm was punched out of the apical part of each ear and for each group the weight of the pooled punches was determined in order to obtain an indication of possible skin irritation.

On study day five (about 66 to 72 hours after the last application of test substance to the ears) the mice were injected intravenously with 20 µCi of 3H-thymidine in 250 µl of sterile saline into a tail vein.

The animals were sacrificed on study day 5 about 5 hours after 3H-thymidine injection by cervical dislocation.

Immediately after the death of each animal a circular piece of tissue (diameter 0.8 cm) was punched out of the apical part of each ear of all animals. The weight of the pooled punches was determined for each test group. These measurements serve for detecting a potential inflammatory ear swelling.

Immediately after removal of the ear punches the left and right auricular lymph nodes were dissected. The weight of the pooled lymph nodes from both sides was determined for each animal.

A concurrent positive control (reliability check) with a known sensitizer was not included into this study. Studies using the positive control substance Alpha-Hexylcinnamaldehyde, techn. 85% are performed twice a year in the laboratory in order to show that the test system is able to detect sensitizing compounds under the test conditions chosen.

Parameter:

SI

Test group / Remarks:

other than limit concentration

Remarks on result:

not measured/tested

Remarks:

only one concentration was tested

Key result

Parameter:

SI

Value:

1.61

Test group / Remarks:

60% in 1% aqueous Pluronic

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: at 60% w/w test substance 1542.7

Test group	Treatment	Cell Count Stimulation Index	³H-thymidinge incorporation Stimulation Index	Lymph Node Weight Stimulation Index	Ear Weight Stimulation Index
1	Vehicle 1% aqueous Pluronic	1.00	1.00	1.00	1.00
2	60% in 1% aqueous Pluronic	1.21	1.61	1.04	1.20

 

No signs of systemic toxicity were noticed.

When applied as 60% preparation in 1% aqueous Pluronic®, the test substance did not induce a biologically relevant response (no increase to 1.5 fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts. There was no relevant increase in lymph node weights, as well.

Reference 3

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 25 MAY 2010 to 30 JUL 2010

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)

Remarks:

according to German Chemical Law and OECD Principles of GLP

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: OP1
- Expiration date of the lot/batch: October 16, 2014
- Certificate of analysis: N° 1873, 15/04/2010

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability in solvent: Stable in water at room temperature
- Storage: At room temperature

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 15 - 25 g
- Housing:single caging in macrolon type II cages with wire mesh top and granulated soft bedding
- Diet: pelleted standard diet (Harlan Laboratories GmbH, 33178 Borchen), ad libitum
- Water: tap water, (Gemeindewerke, 64380 Rossdorf), ad libitum
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness will be used for the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): 45-65%
- Photoperiod (hrs dark / hrs light): artificial light 6:00 a.m. - 6:00 p.m.

Vehicle:

other: ethanol:water (3+7)

Concentration:

10, 25, and 50%
The vehicle and concentration was chosen on the basis of a solubility experiment according to the recommendations in OECD TG
429. The highest test item concentration, which can be technically used was a 50 % (w/w) solution in ethanol:water (3+7).

No. of animals per dose:

4

Details on study design:

In order to study a possible allergenic potential of the test item, three groups each of four female mice were treated with different concentrations of the test item by topical application at the dorsum of each ear (left and right) on three consecutive days. A control group of four mice was treated with the vehicle only. Five days after the first topical application, the mice were intravenously injected into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed and the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a beta-scintillation counter. After the lymph nodes have been excised, both ears of mice were punched at the apical area (Ø 8 mm corresponding to 0.5 cm2). For each animal the weight of both punches was determined.

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated in the body weight tables.

Positive control results:

Experiment performend in May 2010, 5, 10, and 25% alpha-Hexylcinnamaldehyde yielded a S.I. of 2.04, 3.41, and 6.14, respectively. The EC3 value calculated was 8.5%

Key result

Parameter:

SI

Value:

1.61

Test group / Remarks:

10% test item

Key result

Parameter:

SI

Value:

1.64

Test group / Remarks:

25% test item

Key result

Parameter:

SI

Value:

1.64

Test group / Remarks:

50% test item

Test item concentration % (w/w)	Group	Measurement DPM	Calculation			Result
			DPM-BG^a)	number of lymph nodes	DPM per lymph node^b)	S.I.
---	BG I	19	---	---	---	---
---	BG II	44	---	---	---	---
---	1	2249	2218	8	277.2
10	2	3602	3571	8	446.3	1.61
25	3	3666	3635	8	454.3	1.64
50	4	3675	3644	8	455.4	1.64

BG =  Background (1 ml 5% trichloroacetic acid) in duplicate 1=Control Group 2-4=Test Groups

S.I. =  Stimulation Index ^a)= The mean value was taken from the figures BG I and BG II ^b)= Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

The EC3 value could not be calculated, since all S.I.´s are below 3.

Viability / Mortality: No deaths occurred during the study period.

Clinical Signs: No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

Body Weights: The body weight of the animals, recorded prior to the first application and prior to treatment with³HTdR, was within the range commonly recorded for animals of this strain and age.

Ear Weights: The measured ear weights of all animals treated were recorded after necropsy. A statistically relevant increase in ear weights was not observed.

Animal No.	Test Item Concentration % (w/w)	Ear Weights after Necropsy
		Ear Weight (two ears per animal, one punch per ear in mg)	Mean Ear Weight (mg per group)	SD
1	0	25.61	26.06	1.00
2		26.71
3		24.87
4		27.05
5	10	26.71	26.48	0.36
6		26.77
7		26.44
8		25.99
9	25	24.42	25.86	1.03
10		25.97
11		26.85
12		26.19
13	50	25.49	26.68	1.03
14		26.20
15		27.25
16		27.78

Interpretation of results:

GHS criteria not met

Conclusions:

Tested in this study according to OECD test guideline no. 429 and GLP conditions the test item was not a skin sensitizer.

Executive summary:

Three groups each of four female mice were treated daily with the test item at concentrations of 10, 25, and 50% (w/w) in ethanol:sterile water (3+7) by topical application to the dorsum of each ear (left and right) for three consecutive days. A control group of four mice was treated with the vehicle (ethanol:sterile water (3+7)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (³H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of³H-methyl thymidine measured in ab-scintillation counter.

All treated animals survived the scheduled study period and no signs of toxicity were observed. A statistically relevant increase in ear weights was not observed.

A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in 3-fold or greater increase in incorporation of³HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.

In this study Stimulation Indices of 1.61, 1.64, and 1.64 were determined with the test item at concentrations of 10, 25, and 50% in ethanol:sterile water (3+7). The EC3 value could not be calculated, since none of the tested concentrations induced an S.I. greater than 3. The test item was not a skin sensitiser in this assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Two skin sensitisation tests performed according to OECD guideline 429 (LLNA) are available.

In the study from Clariant (1342801, 2010) three groups each of four female mice were treated daily with the test item at concentrations of 10, 25, and 50% (w/w) in ethanol:sterile water (3+7) by topical application to the dorsum of each ear (left and right) for three consecutive days. A control group of four mice was treated with the vehicle (ethanol:sterile water (3+7)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (³H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of³H-methyl thymidine measured in ab-scintillation counter.

All treated animals survived the scheduled study period and no signs of toxicity were observed. A statistically relevant increase in ear weights was not observed.

A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in 3-fold or greater increase in incorporation of³HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.

In this study Stimulation Indices of 1.61, 1.64, and 1.64 were determined with the test item at concentrations of 10, 25, and 50% in ethanol:sterile water (3+7). The EC3 value could not be calculated, since none of the tested concentrations induced an S.I. greater than. The test item was not a skin sensitiser in this assay.

In the BASF study (58H0300/092078, 2010), groups of 5 female CBA/J mice each were treated with a 60% w/w preparation of the test substance in 1% aqueous Pluronic® or with the vehicle alone. The study was carried out as a limit test, using 1 test group and 1 control group. Each test animal was applied with 25 µL per ear of the test substance preparation to the dorsum of both ears for three consecutive days. The control group was treated with 25 µL per ear of the vehicle alone.
Three days after the last application the mice were injected intravenously with 20 µL of ³H-thymidine in 250 µL of sterile saline into a tail vein. About 5 hours after the ³H-thymidine injection, the mice were sacrificed and the auricular lymph nodes were removed. The weights of each animal’s pooled lymph nodes were determined. Thereafter lymph nodes were pooled group wise and further evaluated by measuring their cellular content and ³H-thymidine incorporation into the lymph node cells (indicators of cell proliferation). Moreover, a defined area with a diameter of 0.8 cm was punched out of the apical part of each ear and for each group the weight of the pooled punches was determined in order to obtain an indication of possible skin irritation.
On study day five (about 66 to 72 hours after the last application of test substance to the ears) the mice were injected intravenously with 20 µCi of 3H-thymidine in 250 µl of sterile saline into a tail vein. The animals were sacrificed on study day 5 about 5 hours after 3H-thymidine injection by cervical dislocation. Immediately after the death of each animal a circular piece of tissue (diameter 0.8 cm) was punched out of the apical part of each ear of all animals. The weight of the pooled punches was determined for each test group. These measurements serve for detecting a potential inflammatory ear swelling. Immediately after removal of the ear punches the left and right auricular lymph nodes were dissected. The weight of the pooled lymph nodes from both sides was determined for each animal.

No signs of systemic toxicity were noticed. When applied as 60% preparation in 1% aqueous Pluronic®, the test substance did not induce a biologically relevant response (no increase to 1.5 fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts. There was no relevant increase in lymph node weights, as well.

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 sensitisation, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.

If the formaldehyde content is at or above 0.2 % classification and labelling with Skin Sens. 1, H317 is warranted due to the specific concentration limit of formaldehyde concerning skin sensitisation (Skin Sens. 1; H317: C ≥ 0.2%).