N,N''-(methylenedi-4,1-phenylene)bis[N'-octyl]urea

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

Study period:

November 26, 2002 - December 23, 2002

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

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approx. 9 weeks old
- Weight at study initiation: mean body weight was 18.0 ± 2.1 g.
- Housing: The animals were housed individually in disposable crystal polystyrene cages (22.00 cm x 8.50 cm x 8.00 cm). Each cage contained autoclaved sawdust (SICSA, Alfortville, France).
- Diet: Free access to A04 C pelleted diet (UAR, Villemoisson, Epinay-sur-Orge, France)
- Water: Free access to tap water (filtered using a 0.22 micron filter)
- Acclimation period: at least 5 days before the start of treatment

Sawdust is analysed by the supplier for composition and contaminant levels. Each batch of food is analysed by the supplier for composition and contaminant levels. Bacteriological and chemical analyses of water, including the detection of possible contaminants (pesticides, heavy metals and nitrosamines) are performed regularly by external laboratories. The results of these analyses are archived at CIT.

No contaminants were known to have been present in the diet, drinking water or bedding material at levels which may be expected to have interfered with or prejudiced the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 ºC
- Humidity (%): 30 - 70%
The temperature and relative humidity recorded in the animal room was sometimes outside of the target ranges specified in the study plan. This minor deviation was not considered to have compromised the validity or integrity of the study.
- Air changes (per hr): approximately 12 (filtered, non-recycled air)
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 26 November 2002 to 23 December 2002

Vehicle:

dimethylformamide

Concentration:

Preliminary test: concentrations of 0.5, 1, 2.5 and 5%
Main test: 0, 0.25, 0.5, 1, 2.5 and 5%

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS: The test item was prepared at the concentrations of 0.5, 1, 2.5 and 5%. For three consecutive days, four animals received applications of 25 µL of the dosage form preparations to the external surface of both ears (one concentration per ear). Measurement of the ear thickness (using a micrometer) was performed each day before treatment and 24 hours after the last application.

MAIN STUDY
ANIMAL ASSIGNMENT TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: The test item was considered as a skin sensitizer when the SI for a dose group is ≥ 3. Other relevant criteria such as cellularity, radioactivity levels and ear thickness were also taken into account for the interpretation of results.

TREATMENT PREPARATION AND ADMINISTRATION:
Test substance preparation: The test item was mixed with the required quantity of vehicle and then sonicated for 30 minutes at the temperature of 30 ºC. All dosage form preparations were made freshly on the morning of administration and any unused material was discarded that same day.
Rationale for vehicle: Due to the unsatisfactory solubility of the test item in the first recommended vehicle (acetone/olive oil (4:1, v/v), dimethylformamide was chosen among the other proposed vehicle. A homogeneous dosage preparation was obtained at the maximum concentration of 5%.

Induction - Days 1, 2 and 3:
A dose volume of 25 μL of the control or dosage form preparations was applied to the dorsal surface of both ears, using a adjustible pipette fitted with a plastic tip. In order to avoid licking and to ensure an optimized application of the test materials, the animals were placed under light isoflurane anesthezia during the administration. No massage was performed but the tip was used to spread the preparation over the application sites. No rinsing was performed between each application.

Excision of the nodes - Day 6:
All animals of all groups received a single intravenous injection of 250 µL of 0.9% NaCl containing 20 μCi of 3H-TdR (specific activity of 25 Ci/mmol) via the tail vein. Approximately 5 hours later, the animals were killed by cervical dislocation and the auricular lymph nodes were excised. The lymph nodes were pooled for each experimental group.

Tissue processing for radioactivity - Day 6:
For each experimental group, a single cell suspension of auricular lymph node cells (ALNC) was prepared by mechanical dissagregation in Petri dishes with the plunger of a syringe. Cell suspensions were washed with 15 mL of 0.9% NaCl and pellets obtained were re-suspended in 0.9% NaCl for numeration of lymphocytes (cellularity) and determination of their viability by exclusion of trypan blue. Each cell suspension was then centrifuged and pellets were precipitated with 3 mL of 5% (w/v) trichloroacetic acid (TCA) in purified water at 4ºC overnight.

Radioactivity measurements - Day 7
After a last centrifugation, the pellets were precipitated with 1 mL of 5% TCA. Three mL of Ultima Gold scintillation fluid (Packard) were added in order to measure incorporation of 3H-TdR using β-scintillation counting. The results were expressed as disintegrations/mn (dpm) per group.

Observations:
Clinical signs, morbidity and mortality: The animals were observed at least once a day during the study.
Body weights: The animals were weighed individually on the first day of the study (day 1) and on the day of sacrifice (day 6).
Ear thickness measurements and recording of local reactions: On days 1, 2 and 3 (before application) and on day 6 (after sacrifice), the thickness of the left ear of each animal of the vehicle control and treated groups was measured using a micrometer. No measurement of ear thickness was carried out for animals of the reference treated group.
Any irritation reaction (erythema and oedema) was recorded in parallel. Any other observation (coloration, presence of residual test item,) was noted.

Positive control substance(s):

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

Statistics:

Not performed.

Positive control results:

In the positive control group give HCA at the concentration of 25%, a moderate increase in cellularity and stimulation index exceeding the threshold value of 3 (SI = 15.00) were noted. The study was therefore considered valid.

Key result

Parameter:

SI

Value:

1.17

Test group / Remarks:

0.25%

Key result

Parameter:

SI

Value:

1.06

Test group / Remarks:

0.5%

Key result

Parameter:

SI

Value:

1.52

Test group / Remarks:

1%

Key result

Parameter:

SI

Value:

0.68

Test group / Remarks:

2.5%

Key result

Parameter:

SI

Value:

1.21

Test group / Remarks:

5%

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

Mean DPM/animal values for the experimental groups treated with test substance concentrations 0.25, 0.5, 1, 2.5 and 5% were 673, 607, 871, 388 and 695 DPM respectively. The mean DPM/animal values for the vehicle control and positive control group were 574 and 8616 DPM respectively.

Results Pre-screen test:

The test item was non-irritant in the preliminary test, whatever the concentration. The highest concentration retained for the main test was therefore the maximal practicable concentration, according to the criteria specified in the International Guidelines.

Other results - main study:

Dryness of the skin was noted on day 6 in 1/4 animals at the concentration of 2.5% and in 1/4 animals at the concentration of 5%. Residual test item was recorded on day 3 in all animals of the 5% treated group.

 

A slight increase in ear thickness (+ 20.65%) was noted in the animals given the test item at the concentration of 5%, showing the slight irritant potential of the test item at this concentration. No noteworthy increase in ear thickness was observed in the animals of the other treated groups.

 

The body weight gain of the treated animals was similar to that of the control animals.

    

No clinical signs and no mortality were observed during the study.

The quantity of cells obtained in each group was satisfactory. The cell viability was higher than 80% in each group.

In the treated groups, the incorporation of 3H-TdR was similar to that of the vehicle control group.

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Assessment of contact hypersensitivity to KY-EU in the mouse (Local Lymph Node Assay, 4 females/dose) was conducted according to OECD 429 guidelines and GLP principles.

Since there was no indication that the test substance elicits an SI ≥ 3 when tested up to and including 5%, KY-EU is considered not to be a skin sensitizer.

Based on these results, KY-EU needs not to be classified and has no obligatory labelling requirement for sensitization by skin contact according to Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.

Executive summary:

In a mouse lymph node assay, performed according to OECD test guidelines, mouse were treated with 0, 0.25, 0.5, 1, 2.5 and 5% of KY-EU on three consecutive days by open application on the ears. Three days after the last exposure all animals were injected with 3H-methyl thymidine. Radioactivity measurements of the lymph nodes were performed.

Dryness of the skin was noted on day 6 in 1/4 animals at the concentration of 2.5% and in 1/4 animals at the concentration of 5%. Residual test item was recorded on day 3 in all animals at the concentration of 5%.

A slight increase in ear thickness (+ 20.65%) was noted in the animals given the test item at the concentration of 5%, showing the slight irritant potential of the test item at this concentration. No noteworthy increase in ear thickness was observed in the animals of the other treated groups.

The body weight gain of the treated animals was similar to that of the control animals. 

No clinical signs and no mortality were observed during the study.

The SI values calculated for the substance concentrations 0.25, 0.5, 1, 2.5 and 5% were 1.17, 1.06, 1.52, 0.68 and 1.21 respectively. Since there was no indication that the test substance elicits an SI ≥ 3 when tested up to 5%, KY-EU was considered not to be a skin sensitizer.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In a mouse lymph node assay, performed according to OECD test guidelines, mouse were treated with 0, 0.25, 0.5, 1, 2.5 and 5% of KY-EU on three consecutive days by open application on the ears. Three days after the last exposure all animals were injected with 3H-methyl thymidine. Radioactivity measurements of the lymph nodes were performed.

Dryness of the skin was noted on day 6 in 1/4 animals at the concentration of 2.5% and in 1/4 animals at the concentration of 5%. Residual test item was recorded on day 3 in all animals at the concentration of 5%.

A slight increase in ear thickness (+ 20.65%) was noted in the animals given the test item at the concentration of 5%, showing the slight irritant potential of the test item at this concentration. No noteworthy increase in ear thickness was observed in the animals of the other treated groups.

The body weight gain of the treated animals was similar to that of the control animals.  

No clinical signs and no mortality were observed during the study.

The SI values calculated for the substance concentrations 0.25, 0.5, 1, 2.5 and 5% were 1.17, 1.06, 1.52, 0.68 and 1.21 respectively. Since there was no indication that the test substance elicits an SI ≥ 3 when tested up to 5%, KY-EU was considered not to be a skin sensitizer.

Migrated from Short description of key information:
The skin sensitisation study was performed according to OECD Guideline 429 and GLP principles. Based on the results of the study, KY-EU was considered not to be a skin sensitizer.

Justification for selection of skin sensitisation endpoint:
One in vivo study available.

Respiratory sensitisation

Link to relevant study records

Reference

Endpoint:

respiratory sensitisation

Type of information:

other: expert statement

Adequacy of study:

weight of evidence

Study period:

March 2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Expert evaluation based on the available information on respiratory sensitisation for KY-EU; acceptable for assessment purposes.

Qualifier:

no guideline required

Principles of method if other than guideline:

Expert statement from a toxicologist, based on the evaluation of available information on KY-EU.

GLP compliance:

no

Results:

Literature search for KY-EU and the three individual components of KY-EU using SciFinder databases (CAPLUS and MedLine) did not result in any relevant publication related to respiratory sensitisation. KY-EU related respiratory sensitization is also not reported in medical reports of workers in KY-EU plants, as provided by an occupational hygienists (medical doctor).

For the endpoint respiratory sensitisation there are no formally recognized and validated animal tests, as indicated in the ECHA guidance on information requirements and guidance on application of the CLP criteria. However, data from some animal studies may be indicative of the potential of a substance to cause respiratory sensitisation in humans and may provide supportive evidence in case human evidence is available. All available data in the dossier for KY-EU has been evaluated for relevant information.
Relevant human information with respect to respiratory sensitisation may be available from case reports, epidemiological studies, medical surveillance or reporting schemes. Respiratory hypersensitivity in humans is normally seen as asthma, rhinitis/conjunctivitis or alveolitis. For KY-EU no human data on respiratory sensitisation is known, neither from the open literature, nor from medical reports.
Data from animal studies that may be indicative of the potential of a substance to cause sensitisation by inhalation in humans may include measurements of specific immunological parameters in mice (e.g. IgE) or specific pulmonary responses in guinea pigs. No such data are available for KY-EU, neither from the open literature nor from the available data in the registration dossier (IUCLID).
In a way of evidence, information on skin sensitizing activity of substances can also be taken into consideration, as there may be a relationship between skin sensitizing properties of a substance and respiratory sensitizing properties. The interpretation is that a substance which fails to induce a positive response in the LLNA (at an appropriate test concentration and with the exception of large substances such as enzymes) most probably lacks the potential for respiratory allergy.
Following the integrated evaluating strategy for respiratory data, as presented in ECHA endpoint specific guidance R.7a (figure R.7.3-2), it is demonstrated that a substance that lacks skin sensitising properties is not a respiratory sensitiser. Chemicals that act through non-immunological mechanisms are usually identified on the basis of evidence from human exposure. [3] KY-EU is not a skin sensitiser.
Moreover, DEREK NEXUS analysis for all 3 components of KY-EU demonstrated no alerts for the endpoint respiratory sensitization.

Interpretation of results:

not sensitising

Conclusions:

Based on the evaluation of all available information on respiratory sensitisation for KY-EU (open literature, all toxicity data, QSAR prediction, medical stetement) it is concluded that KY-EU is considered to have no repiratory sensitising properties.

Executive summary:

To evaluate the potential respiratory sensitising properties of KY-EU, a literature search was performed (including search in international databases for chemicals) and screened for information on respiratory sensitisation related to KY-EU), prevalance of respiratory diseases amongst workers with KY-EU recorded by an occupational physician, evaluation of all toxicity data from KY-EU and a QSAR prediction by DEREK Nexus were considered.

In the open literature there is no information, neither on human nor on animal, related to respiratory sensitising properties of KY-EU or its individual components. In addition, also medical reports of plant workers provided by health doctors do not indicate respiratory sensitising properties of KY-EU, as shown by absence of reports related to respiratory sensitising complaints of workers (period 2004-2015)

In the absence of any relevant information on respiratory sensitizing properties of KY-EU and its individual components, both from human and animal reports, the integrated evaluating strategy for respiratory sensitisation data according to ECHA endpoint specific data is applicable. As KY-EU has no skin sensitizing properties as demonstrated by a negative LLNA test, and there are no human data indicating respiratory sensitising properties (by means of immunological or non-immunological mechanisms), this substance is not a respiratory sensitiser as presented in the REACH guidance scheme R7A, figure 7.3-2. [3] This is also confirmed by DEREK NEXUS prediction for the endpoint respiratory sensitization: all 3 components of KY-EU demonstrated to fire no alert for respiratory sensitization. The substantiation for listing KY-EU as respiratory sensitiser is unclear. With all available information on KY-EU and its individual components there is no indication for respiratory sensitising properties of KY-EU.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The substance is listed in Annex VI of the CLP Regulation (01 ATP, Index No 006 -103 -00 -7) and should be labelled for respiratory sensitisation with: May cause allergy or asthma symptoms or breathing difficulties if inhaled (H334).

In the open literature there is no information, neither on human nor on animal, related to respiratory sensitising properties of KY-EU or its individual components. In addition, also medical reports of plant workers provided by health doctors do not indicate respiratory sensitising properties of KY-EU, as shown by absence of reports related to respiratory sensitising complaints of workers (period 2004-2015)

In the absence of any relevant information on respiratory sensitizing properties of KY-EU and its individual components, both from human and animal reports, the integrated evaluating strategy for respiratory sensitisation data according to ECHA endpoint specific data is applicable. As KY-EU has no skin sensitizing properties as demonstrated by a negative LLNA test, and there are no human data indicating respiratory sensitising properties (by means of immunological or non-immunological mechanisms), this substance is not a respiratory sensitiser as presented in the REACH guidance scheme R7A, figure 7.3-2. [3] This is also confirmed by DEREK NEXUS prediction for the endpoint respiratory sensitization: all 3 components of KY-EU demonstrated to fire no alert for respiratory sensitization. The substantiation for listing KY-EU as respiratory sensitiser is unclear. With all available information on KY-EU and its individual components there is no indication for respiratory sensitising properties of KY-EU.

Migrated from Short description of key information:
The substance is listed in Annex VI of the CLP Regulation (01 ATP, Index No 006 -103 -00 -7) with the classification Resp. Sens., Cat. 1. However, the substantiation for the current Annex VI classification of the substance as respiratory sensitiser is unclear. With all available information on KY-EU and its individual components there is no indication for respiratory sensitising properties of KY-EU. A CLH proposal to remove the current Annex VI classification of the substance as respiratory sensitiser will therefore be prepared.

Justification for selection of respiratory sensitisation endpoint:
Expert statement considering all available relevant information for the endpoint respiratory sensitisation, including medical statement, scientific literature and QSAR prediction.

Justification for classification or non-classification

Based on the available negative result in a LLNA study, the substance KY-EU does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.

The substance is listed in Annex VI of the CLP Regulation (01 ATP, Index No 006 -103 -00 -7) with the classification Resp. Sens., Cat. 1 and should be labelled for respiratory sensitisation with: May cause allergy or asthma symptoms or breathing difficulties if inhaled (H334). The justification for this is unclear. With all available information on KY-EU and its individual components there is no indication for respiratory sensitising properties of KY-EU.