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Description of key information

A guinea pig study carried out with Monuron (33% water paste) applied to both intact and abraded skin of male albino guinea pigs did not result in allergic skin sensitization. However, as only limited details were available, the study was considered as supporting data. Weight-of-evidence was therefore used from read-across substance Fenuron (see attached read-across justification): First, Fenuron revealed a mean cysteine and lysine peptide depletion of 2.580% and, hence, the test item is considered negative and predicted to be a non-sensitiser (no or minimal reactivity) in the Direct Peptide Reactivity Assay (DPRA). Secondly, Fenuron was confirmed negative for sensitising properties in the ARE-Nrf2 luciferase test method.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in chemico
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
see read across justification attached to section 13
Reason / purpose for cross-reference:
read-across source
Positive control results:
Cinnamic aldehyde was used as positive control at a concentration of 100 mM in acetonitrile. Treatment with the positive control item revealed a cysteine and lysine peptide depletion of 70.81% for cysteine and 51.15% for lysine peptide. These values are within the required range of 60.8% and 100% for the cysteine peptide and between 40.2% and 69.0% for the lysine peptide. The maximum standard deviation (SD) for the positive control replicates were < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion. Therefore, the study can be regarded as valid.
Run / experiment:
other: Cysteina+ Lysine
Parameter:
other: % depletion
Value:
2.58
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
Reactivity Class (Cysteine+ Lysine) = Negative / Reactivity Class (Cysteine only) = Negative
Other effects / acceptance of results:
Two reference controls containing only 0.5 mM cysteine peptide solution or 0.5 mM lysine peptide solution and acetonitrile were also included in the HPLC run sequence and were used to verify the HPLC system suitability prior to analysis (reference controls A) and the stability of the reference controls over time (reference control B). To verify that the solvent used to dissolve the test item does not impact the percent peptide depletion the reference control C was prepared by adding acetonitrile (vehicle) to the peptide solution. The reference control C was used to calculate the percent peptide depletion for the test item. Each sample was tested in triplicate.
No precipitate in the reaction mixture at the end of the incubation time and no coelution were observed.
IsoQure UR 300-treated samples revealed a cysteine peptide depletion of 3.02% and a lysine peptide depletion of 2.14% (mean peptide depletion of 2.580%) and, hence, were well below 6.38%. IsoQure UR 300 is considered negative and predicted to be a non-sensitiser (no or minimal reactivity) in the Direct Peptide Reactivity Assay (DPRA).
Cinnamic aldehyde was used as positive control at a concentration of 100 mM in acetonitrile. Treatment with the positive control item revealed a cysteine and lysine peptide depletion of 70.81% for cysteine and 51.15% for lysine peptide. These values are within the required range of 60.8% and 100% for the cysteine peptide and between 40.2% and 69.0% for the lysine peptide. The maximum standard deviation (SD) for the positive control replicates were < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion. Therefore, the study can be regarded as valid.

The linearity of the standard calibration curve was r2 = 1.0000 for cysteine peptide and r2 = 0.9999 for lysine peptide. Hence the requirement of r2 > 0.99 was met.
The mean peptide concentrations of reference control A were 0.508 or 0.494 mM cysteine or lysine peptide, respectively and, hence well within the accepted range of 0.50 ± 0.05 mM and the coefficient of variation (CV) of peptide peak areas for the nine reference controls B and C was <15.0%.

The mean peptide concentrations of reference control A were 0.508 or 0.494 mM cysteine or lysine peptide, respectively and, hence well within the accepted range of 0.50 ± 0.05 mM and the coefficient of variation (CV) of peptide peak areas for the nine reference controls B and C was <15.0%.
Interpretation of results:
study cannot be used for classification
Conclusions:
Read-across tets item IsoQure UR 300 revealed a mean cysteine and lysine peptide depletion of 2.580% and, hence, the read-across test item is considered negative and predicted to be a non-sensitiser (no or minimal reactivity) in the Direct Peptide Reactivity Assay (DPRA).
Executive summary:

The purpose of this study was to determine the sensitising potential of read-across test item IsoQure UR 300 in a Direct Peptide Reactivity Assay (DPRA). The study was performed according to OECD guideline 442C. The DPRA is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 ± 2 hours incubation with the test item at 25 ± 2.5°C. Relative peptide concentration is measured by high-performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 nm. Cysteine and lysine peptide percent depletion values are then calculated and used in a prediction model, which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers. The read-across test item was dissolved at a concentration of 100 mM in acetonitrile. Two reference controls containing only 0.5 mM cysteine peptide solution or 0.5 mM lysine peptide solution and acetonitrile were also included in the HPLC run sequence and were used to verify the HPLC system suitability prior to analysis (reference controls A) and the stability of the reference controls over time (reference control B). To verify that the solvent used to dissolve the test item does not impact the percent peptide depletion the reference control C was prepared by adding acetonitrile (vehicle) to the peptide solution. The reference control C was used to calculate the percent peptide depletion for the test item. Each sample was tested in triplicate. IsoQure UR 300-treated samples revealed a cysteine peptide depletion of 3.02% and a lysine peptide depletion of 2.14% (mean peptide depletion of 2.580%) and, hence, were well below 6.38%. Read-across test item IsoQure UR 300 is considered negative and predicted to be a non-sensitiser (no or minimal reactivity) in the Direct Peptide Reactivity Assay (DPRA). Cinnamic aldehyde was used as positive control at a concentration of 100 mM in acetonitrile. Treatment with the positive control item revealed a cysteine and lysine peptide depletion of 70.81% for cysteine and 51.15% for lysine peptide. These values are within the required range of 60.8% and 100% for the cysteine peptide and between 40.2% and 69.0% for the lysine peptide. The maximum standard deviation (SD) for the positive control replicates were < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion. Therefore, the study can be regarded as valid.

The acceptance criteria of validity were fulfilled in this test.

Endpoint:
skin sensitisation: in vitro
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
see read across justification attached to section 13
Reason / purpose for cross-reference:
read-across source
Positive control results:
The positive control cinnamic aldehyde was run in both repetitions. The detailed results for the positive control are reported needs to be positive for a run to be accepted (i.e. induction > 1.5 fold). This was the case in both repetitions. The induction at 64 µM and the EC1.5 for cinnamic aldehyde were also calculated. The targets are:
• the average induction in the two replicates for cinnamic aldehyde at 64 µM should be between 2 and 8,
• the EC1.5 value should be within two standard deviations of the historical mean value .
At least one of these two numerical criteria must be met in order to accept a repetition. In the experiments performed both criteria were fulfilled in both repetitions.Thus both repetitions were valid for the positive control.
In addition, the EC1.5 average value of the positive control of 19.05 µM is within two standard deviations of the historical mean .
Run / experiment:
other: Luciferase determinations
Parameter:
other: Imax
Value:
1.35
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not applicable
Run / experiment:
other: Luciferase determinations
Parameter:
other: EC1.5 [µM]
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: no concentration with calculated ≥ 1.5 fold luciferase induction
Run / experiment:
other: Cytotoxicity determinations
Parameter:
other: IC50 [µM]
Value:
2 000
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Run / experiment:
other: Cytotoxicity determinations
Parameter:
other: IC30 [µM]
Value:
1 366.16
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Other effects / acceptance of results:
The maximum average fold induction of the luciferase activity (Imax) value observed at any concentration of the test item was 1.35 ± 0.28 and no EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) could be calculated.
The calculated IC50 value was > 2000 µM IsoQure UR 300 and the IC30 was 1366.16 µM for 30% reduction of cellular viability, respectively.
The KeratinoSensTM prediction of the test item is considered negative as the luciferase induction value was < 1.5 compared to the solvent control.
As further performance criterion the variability of the luminescence reading for the negative control DMSO must be below 20% in each repetition which consists of 6 wells tested in triplicate. The the average coefficients of variation (CV) for the negative control were 9.32% or 13.07% for the first or second repetition, respectively.
Interpretation of results:
study cannot be used for classification
Conclusions:
Read-across test item IsoQure UR 300 revealed no sensitising properties in the ARE-Nrf2 Luciferase test method.
Executive summary:

Read-across test item IsoQure UR 300 was examined for sensitising properties in theARE-Nrf2 luciferase test method. The ARE-Nrf2 luciferase test method makes use of an immortalised adherent cell line derived from HaCaT human keratinocytes stably transfected with a selectable plasmid. The cell line contains the luciferase gene under the transcriptional control of a constitutive promoter fused with an ARE element from a gene that is known to be up-regulated by contact sensitisers. The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes, and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic test substances. Two endpoints were measured: luciferase induction after a 48 hour treatment with IsoQure UR 300 and cytotoxicity determined with the MTT assay with the same cell batch and employing the same dilutions of the test item. DMSO was used as solvent control. For Luciferase induction the maximal fold-induction over solvent control (Imax) and the concentration needed to reach an 1.5 fold induction (EC1.5) were calculated. For cytotoxicity the IC50 and IC30 values were interpolated. IsoQure UR 300 was completely dissolved in dimethyl sulfoxide (DMSO). DMSO was employed as negative control. IsoQure UR 300was tested at 12 concentrations in the range from 0.98 to 2000 µM. Cinnamic aldehyde tested at five concentrations from 4 – 64 µM was used as the positive control. Two independent repetitions with three parallel technical replicates were run with this same set-up, and one parallel plate was prepared for cytotoxicity determination. For the MTT data the % viability was then calculated for each well in the test plate in relation to average of the six solvent control wells. For the luciferase data the average value of the six solvent control wells was set to 1, and for each well in the test plate the fold induction was calculated in relation to this value. The maximum average fold induction of the luciferase activity (Imax) value observed at any concentration of the test item was1.35 ± 0.28 and no EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) could be calculated .The calculated IC50 value was > 2000µM IsoQure UR 300 and the IC30 was1366.16 µM for 30% reduction of cellular viability, respectively. The KeratinoSensTM prediction of the read-across test item is considered negative as the luciferase induction value was < 1.5 compared to the solvent control. The solvent control and the positive control cinnamic aldehyde were run in both repetitions. All quality criteria for luciferase induction and variability of the solvent control and positive control required were fulfilled.

In conclusion, read-across test item IsoQure UR 300 revealed no sensitising properties in the ARE-Nrf2 Luciferase test method.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

A guinea pig study was carried out to evaluate the primary irritation and skin sensitization potential of Monuron (Zapp, 1955). A 33% water paste of Monuron was applied to both intact and abraded skin of male albino guinea pigs. The compound was practically non irritating and did not cause allergic skin sensitization. No further details were given on such parameters as numbers used or period of experimentation. The author concluded that a moderate amount of skin contact with Monuron would not be harmful because it is not an irritant or a sensitizer, nor is it significantly absorbed through the skin. However, as only limited details were available, the study was considered as supporting data.

Following weight-of-evidence was also available from the read-across substance Fenuron (see attached read-across justification):

Fenuron was examined for sensitising potential in a Direct Peptide Reactivity Assay (DPRA according to OECD guideline 442C (Rehders, 2018). Cysteine and lysine peptide percent depletion values were calculated and used in a prediction model, which allowed assigning the test item to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers. Fenuron -treated samples revealed a cysteine peptide depletion of 3.02% and a lysine peptide depletion of 2.14% (mean peptide depletion of 2.580%) and, hence, were well below 6.38%. IsoQure UR 300 is considered negative and predicted to be a non-sensitiser (no or minimal reactivity) in the Direct Peptide Reactivity Assay (DPRA).

Fenuron was examined for sensitising properties in the ARE-Nrf2 luciferase test method (Spruth, 2018a). Two endpoints were measured: luciferase induction after a 48 hour treatment and cytotoxicity determined with the MTT assay with the same cell batch and employing the same dilutions of the test item. DMSO was used as solvent control. For Luciferase induction the maximal fold-induction over solvent control (Imax) and the concentration needed to reach an 1.5 fold induction (EC1.5) were calculated. For cytotoxicity the IC50 and IC30 values were interpolated. Fenuron was completely dissolved in dimethyl sulfoxide (DMSO), and DMSO was employed as negative control. Fenuron was tested at 12 concentrations in the range from 0.98 to 2000 µM. Cinnamic aldehyde tested at five concentrations from 4 – 64 µM was used as the positive control. The maximum average fold induction of the luciferase activity (Imax) value observed at any concentration of the test item was 1.35 ± 0.28 and no EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) could be calculated. The calculated IC50 value was > 2000µM Fenuron and the IC30 was 1366.16 µM for 30% reduction of cellular viability, respectively. In conclusion, Fenuron revealed no sensitising properties in the ARE-Nrf2 Luciferase test method.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on these results and according to CLP (No. 1272/2008 of 16 December 2008), Monuron does not have to be classified and has no obligatory labelling requirement for skin sensitisation.