Tetrahydro-1,3-dimethyl-1H-pyrimidin-2-one

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP 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)

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: pre-test 1 and 2: Charles River UK, Manston Road, Margate, Kent CT9 4LT, United Kingdom; pre-test 3 and main experiment: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: pre-test 1: 10-11 weeks; pre-test 2: 11-12 weeks; pre-test 3: 8-9 weeks; main study: 9-10 weeks
- Weight at study initiation: main study: 20.4 ± 0.9 g (mean ± SD)
- Housing: group housing in Makrolon Type II (pre-tests)/ III (main study) cages, with wire mesh top (EHRET GmbH, 79302 Emmendingen, Germany)
- Diet: Pelleted standard diet, ad libitum (Harlan Laboratories B.V., 5960 AD Horst, Netherlands)
- Water: Tap water, ad libitum (Gemeindewerke, 64380 Rossdorf, Germany)
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 30 – 65 (acclimation period); 45 – 65 (main study)
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12 / 12 (6.00 p.m. - 6.00 a.m. / 6.00 a.m. - 6.00 p.m.)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

5, 10 and 25% (w/w)

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which could be technically used, was the undiluted test item (100%). Test item solution at different concentrations was prepared using acetone:olive oil (4+1 v/v) as vehicle.
- Irritation: To determine the highest non-irritant test concentration that does not induce signs of systemic toxicity at the same time, three pre-tests were performed in two animals, each. In the first pre-test, two mice were treated by topical application to the dorsal surface of each ear with test item concentrations of 50% (w/w) and 100% once daily each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local irritation were documented and a score was used to grade a possible reddening of the ear skin. Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer (S0247 Kroeplin, 36381 Schlüchtern, Germany). Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Stiefel, Ø 8 mm corresponding to 0.5 cm²) and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if an erythema of the ear skin of a score value ≥3 was observed at any observation time and/or if an increase in ear thickness of ≥25% was recorded on day 3 or day 6. The measured ear weight was also considered in this evaluation.
On day 2, both animals were found dead at the tested concentrations of 50 and 100%.
Thus, a second pre-test was performed as mentioned above, using test item concentrations of 5 and 10% (w/w). From day 2 to 5, an erythema of the ear skin was observed in both animals (Score 1), but signs of systemic toxicity were not observed.
Thus, a third pre-test was performed using a test item concentration of 25% (w/w). From day 2 to day 6, an erythema of the ear skin was observed in the animal (days 2, 3, 5, and 6: Score 1, day 4: Score 2).
Thus, the test item in the main study was assayed at 5, 10, and 25% (w/w).

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled: First, that exposure to at least one concentration of the test item resulted in an incorporation of ³HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index. Second, that 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 ADMINISTRATION:
The test item was placed into an appropriate container on a tared balance and acetone:olive oil (4+1 v/v) was added to achieve the required test item concentration. The different test item concentrations were prepared individually. The preparations were made freshly before each dosing occasion. Concentrations were in terms of material as supplied. Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with test item concentrations of 5, 10, and 25% (w/w) in acetone:olive oil (4+1 v/v). The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (Ø ~ 8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone.

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, for the ear weights, the lymph node weights and lymph node cell count, and for the DPM values (group mean DPM ± standard deviation).
A statistical analysis was conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between test item groups and negative control group. For all statistical calculations SigmaStat for Windows (Version 2.0) was used. A One-Way-Analysis-of-Variance was used as statistical method. In case of significant results of the One-Way-ANOVA, multiple comparisons were performed with the Dunnett test. Statistical significance was set at the five per cent level (p < 0.05). The Dean-Dixon-Test and Grubb’s test were used for identification of possible outliers (performed with Microsoft Excel 2003).
However, both biological and statistical significance were considered together.

Positive control results:

The periodic positive control experiment was performed with α-hexyl cinnamaldehyde dissolved in acetone:olive oil (4:1 v/v) (compound listed in OECD 429 Guideline) in December 2011. An EC3 value of 14.4% (w/v) was calculated.

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

Stimulation Indices (S.I.) of 0.63, 0.59 and 0.77 were determined with the test item at concentrations of 5, 10, and 25% (w/w) in acetone:olive oil (4+1 v/v), respectively. The EC3 value could not be calculated, since all S.I.'s are below the threshold value of 3.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: A statistically significant or biologically relevant increase in DPM value was not observed in any test group in comparison to the vehicle control group.

Calculation of Stimulation Indices per Dose Group

Test item concentration	Group Calculation	SD	S.I.
	Mean DPM per animal (2 lymph nodes)^a)
Vehicle Control (acetone:olive oil (4+1 v/v))	1407.8	408.6	1.00
25% 2-Ethylhexylchloride	889.0	223.1	0.63
50% 2-Ethylhexylchloride	837.6	279.6	0.59
100% 2-Ethylhexylchloride	1091.0	416.6	0.77

a)  Mean DPM/animal was determined by dividing the sum of the measured values from lymph
     nodes of all animals within a group by the number of animals in that group (5 animals)

 

 

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 with ³HTdR, was within the range commonly recorded for animals of this strain and age.

Lymph Node Weights and Cell Counts: The measured lymph node weights and – cell counts of all animals treated were recorded after sacrifice. A statistically significant or biologically relevant increase in lymph node weights and lymph node cell count was not observed in any of the test item treated groups in comparison to the vehicle control group. For BALB/c mice, a cutoff-value for the lymph node cell count index of 1.55 was reported for a positive response (Ehling G. et al. (2005), Toxicology 212, 69-79). The indices determined for the lymph node cell count did not exceed this threshold.

Ear Weights: A statistically significant increase in ear weights was observed in the low and high dose group in comparison to the vehicle control group. For BALB/c mice, a threshold for the ear weight index of 1.1 was reported for a positive response (Ulrich P. et al. (2001), Archives of Toxicology 74, 733-744). None of the indices determined exceeded this threshold.

 

Table of Ear Weights

Ear Weights after Sacrifice
Test item concentration % (w/w)	Animal No.	Ear weight per animal (mg)	Mean Ear weight (mg)	SD	Index (Value Test Group versus Value Control)
0	1	21.70	23.1	1.0	1.0
	2	23.35
	3	22.90
	4	23.17
	5	24.37
5	6	24.18	26.2*	2.9	1.1
	7	23.57
	8	26.48
	9	31.03
	10	25.69
10	11	23.58	25.2	1.2	1.1
	12	26.34
	13	24.59
	14	25.13
	15	26.32
25	16	25.23	25.9*	0.8	1.1
	17	27.12
	18	26.37
	19	25.19
	20	25.81

Index = mean ear weight of test item group related to vehicle group

*statistically significant increase vs. control group (p<0.05)

 

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item N,N’-Dimethyl propylene urea was not a skin sensitiser under the test conditions of this study.

Executive summary:

The study was performed according to OECD guideline 429 in compliance with GLP.

In this study the test item N,N’-Dimethyl propylene urea wasassessed for its skin sensitising potential using the Local Lymph Node Assay (LLNA) in mice. Test item solution at 5, 10, and 25% (w/w) were prepared in the vehicle acetone:olive oil (4+1 v/v). The highest concentration tested was the highest concentration that could be applied whilst avoiding systemic toxicity and excessive local skin irritation (as determined by three pre-experiments).

The animals did not show any signs of systemic toxicity or local skin irritation during the course of the study and no cases of mortality were observed. A statistically significant increase in ear weights was observed in the low and the high dose group in comparison to the vehicle control group (p<0.05). However, this was considered to be not biologically relevant, as the mean value of the groups was still in the range of historical ear weight control data for the relevant vehicle (range: 23.28 to 27.81 mg). Furthermore, for BALB/c mice, a threshold for the ear weight index of 1.1 was reported for a positive response (Ulrich P. et al. (2001), Archives of Toxicology 74, 733 -744). None of the indices determined exceeded this threshold.

Stimulation Indices (S.I.) of 0.63, 0.59 and 0.77 were determined with the test item at concentrations of 5, 10, and 25% (w/w) in acetone:olive oil (4+1 v/v), respectively. A statistically significant or biologically relevant increase in DPM value and also in lymph node weight and cell count was not observed in any test group in comparison to the vehicle control group. Furthermore, the cutoff-value of 1.55 for a positive response regarding the lymph node cell count index reported for BALB/c mice (Ehling G. et al. (2005), Toxicology 212, 659 -79) was not exceeded in any group. A dose response was also not observed in any of the mentioned parameters.

Conclusion: The test item N,N’-Dimethyl propylene urea was thus not a skin sensitiser under the test conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

To test the sensitizing potential of the test substance N,N’-Dimethyl propylene urea a local lymph node asssay was performed according to OECD guideline 429 and GLP.

Initially, three pre-tests were performed in two animals, each. In the first pre-test, two mice were treated by topical application to the dorsal surface of each ear with test item concentrations of 50% (w/w; using acetone:olive oil (4+1 v/v) as vehicle) and 100% once daily each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local irritation were documented and a score was used to grade a possible reddening of the ear skin. Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer (S0247 Kroeplin, 36381 Schlüchtern, Germany). Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Stiefel, Ø 8 mm corresponding to 0.5 cm²) and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if an erythema of the ear skin of a score value ≥3 was observed at any observation time and/or if an increase in ear thickness of ≥25% was recorded on day 3 or day 6. The measured ear weight was also considered in this evaluation. On day 2, both animals were found dead at the tested concentrations of 50 and 100%. Thus, a second pre-test was performed as mentioned above, using test item concentrations of 5 and 10% (w/w). From day 2 to 5, an erythema of the ear skin was observed in both animals (Score 1), but signs of systemic toxicity were not observed. Thus, a third pre-test was performed using a test item concentration of 25% (w/w). From day 2 to day 6, an erythema of the ear skin was observed in the animal (days 2, 3, 5, and 6: Score 1, day 4: Score 2). Thus, the test item in the main study was assayed at 5, 10, and 25% (w/w) in acetone:olive oil (4+1 v/v). The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (Ø ~ 8 mm) of each ear once daily for three consecutive days.

A statistically significant or biologically relevant increase in DPM value was not observed in any test group in comparison to the vehicle control group. Stimulation Indices (S.I.) of 0.63, 0.59 and 0.77 were determined with the test item at concentrations of 5, 10, and 25% (w/w) in acetone:olive oil (4+1 v/v), respectively. The EC3 value could not be calculated, since all S.I.'s are below the threshold value of 3. The substance is hence classified as not sensitising.

Migrated from Short description of key information:
LLNA (OECD 429): not sensitizing (BASDF SE/Harlan, 2012)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

Justification for selection of respiratory sensitisation endpoint:
In accordance with REACH Annex VIII, the study does not need to be conducted.

Justification for classification or non-classification

In the evaluation of all available information, the test substance is not classified with regard to sensitisation according to Directive 67/548/EEC (DSD) and to Regulation (EC) No 1272/2008 (CLP), respectively.