Pyrazolo[5,1-b]quinazolin-9(1H)-one, 2-methyl-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 October 2014 - 29 October 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

2-methyl-1H,9H-pyrazolo[3,2-b]quinazolin-9-one

EC Number:

616-079-5

Cas Number:

74336-55-3

Molecular formula:

C11 H9 N3 O

IUPAC Name:

2-methyl-1H,9H-pyrazolo[3,2-b]quinazolin-9-one

Details on test material:

- Physical state: Beige solid
- Storage condition of test material: At room temperature

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd (SPF)

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: 1st pre-test: 9 - 10 weeks (beginning of treatment); Main study: 8 - 9 weeks (beginning of treatment)
- Weight at study initiation: average 19.3 g
- Housing: group housing in Makrolon Type II (pre-test) / III (main study) cages
- Diet: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
- Water: tap water, ad libitum
- Acclimation period: At least 5 days prior to the start of dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 45-65%
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

5, 10, and 20%
The highest test item concentration, which could be technically used, was a 20% suspension in PG. Vortexing was used to formulate the test item.

No. of animals per dose:

Number of animals for the pre-test: 2 females
Number of animals per group in the main study: 5 females (nulliparous and non-pregnant)

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 a 20% suspension in PG.
- Irritation: To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed in two animals. Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 10 and 20% once daily each on three consecutive days.
- Lymph node proliferation response: 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. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2) and were immediately pooled per animal and weighed using an analytical balance.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA according to OECD 429
- 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.
Furthermore, an index was calculated for the lymph node weight and –cell count as well as for the ear weight by dividing the mean values of the test item treated groups by the mean of the vehicle treated group. For BALB/c mice, a cut-off value for the lymph node cell count index of 1.55 was reported for a positive response and the cut-off value for the ear weight index regarding a positive response (ear irritation) was reported to be 1.1. However, these cut-off values mentioned in the respective papers have been determined using a different strain of mice and can thus not be implicitly adopted.

TREATMENT PREPARATION AND ADMINISTRATION:
Test Item Preparation:
The test item was placed into an appropriate container on a tared balance and PG was added. The different test item concentrations were prepared individually. Homogeneity of the test item in the vehicle was maintained during treatment using a magnetic stirrer. In the main experiment small clots were observed in all prepared test item concentrations. The preparations were made freshly and used within two hours before each dosing occasion. Concentrations were in terms of material as supplied.
Topical application:
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 5, 10, and 20% in PG. The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (diameter of 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 (control animals).

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).
Where appropriate, the EC3 value was calculated according to the equation EC3 = (a-c) [(3-d)/(b-d)] + c where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.
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 the test item groups and negative control group. For all statistical calculations custom made statistical program ´R` Decision Tree was used. Statistical significance was set at the five per cent level (p < 0.05).
The Dean-Dixon-Test and the Grubb’s test were used for detection of possible outliers (performed with custom made statistical program ´R` Decision Tree).
However, both biological and statistical significance were considered together.

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

2.54

Test group / Remarks:

5% tes substance

Results 2

Parameter:

SI

Value:

2.43

Test group / Remarks:

10% test substance

Results 3

Parameter:

SI

Value:

3.14

Test group / Remarks:

20% test substance

Results 4

Parameter:

EC3

Value:

18

Any other information on results incl. tables

Calculation of Stimulation Indices per Dose Group

	Group Calculation
Test item concentration	Mean DPM per animal (2 lymph nodes)^a)	SD	S.I.
Vehicle Control Group (propylene glycol)	690.1	339.7	1.00
5% test substance	1749.9	838.5	2.54
10% test substance	1674.9	850.6	2.43
20% test substance	2167.3	288.6	3.14

^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)

^$Mean DPM value for the group was according to the ANOVA (Dunnett-test) significantly higher than the corresponding control value. The p value for the analysis was p<0.05)

Calculation of EC3 value

	Test item concentration %	S.I.
Test Group 3	10 (a)	2.43 (b)
Test Group 4	20 (c)	3.14 (d)
EC3 = (a-c) [(3-d)/(b-d)] + c = 18% (w/w)

Clinical Signs

Neither signs of systemic toxicity nor local skin effects 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 and biologically relevant increase in lymph node weights and –cell counts was observed in the high dose group in comparison to the vehicle control group. For BALB/c mice, a cut-off value for the lymph node cell count index of 1.55 was reported for a positive response. The index of the highest dose group determined for the lymph node cell count almost reached this threshold (index of 1.54).

Ear Weights

The measured ear weight of all animals treated was recorded on test day 6 (after necropsy). A statistically significant but not biologically relevant increase in ear weights was observed in the mid and high dose group in comparison to the vehicle control group. The cut-off value (1.1) of the ear weight index for a positive response regarding ear skin irritation reported for BALB/c mice was not exceeded in any of the treated groups.

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

The test item was found to be a skin sensitiser under the test conditions of this study.

Executive summary:

In this study the test item was assessed for its skin sensitising potential using the Local Lymph Node Assay (LLNA) in mice. Test item suspensions at different concentrations were prepared in the vehicle propylene glycol (PG). For this purpose a local lymph node assay was performed using test item concentrations of 5, 10, and 20% (w/w). The highest concentration tested was the highest concentration that could be technically achieved whilst avoiding systemic toxicity and excessive local skin irritation (as determined by a pre-experiment).

The animals showed neither any signs of systemic toxicity nor local skin effects during the course of the study and no cases of mortality were observed. A statistically significant increase in ear weights was observed in the mid and the high dose group in comparison to the vehicle control group (p<0.05). For BALB/c mice, a cut-off value of 1.1 for the ear weight index was reported for a positive response regarding ear skin irritation. None of the indices determined for the test item treated groups exceeded this threshold. Therefore, the statistically increased ear weights were considered to be not biologically relevant.

A test item is regarded as a sensitiser in the LLNA if exposure to one or more test item concentration results in a 3-fold or greater increase in incorporation of ³HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated test item concentration required to produce a S.I. of 3 is referred to as the EC3 value. In this study Stimulation Indices (S.I.) of 2.54, 2.43, and 3.14 were determined with the test item at concentrations of 5, 10, and 20% (w/w) in PG, respectively. A statistically significant and biologically relevant increase in DPM value and also in lymph node weight and cell count was observed in the highest dose group in comparison to the vehicle control group. Furthermore, the cut-off value of 1.55 for a positive response regarding the lymph node cell count index reported for BALB/c mice was almost reached in the highest dose group (index of 1.54), thus corroborating the study result. The test item was found to be a skin sensitizer and an EC3 value of 18% was derived.