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Skin sensitisation

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skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21.09. – 05.10. 2010
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Adopted 22nd July 2010
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Test material form:
Details on test material:
Expiration: 03/2011
Storage condition of test material: at the room temperature in tightly closed container

In vivo test system

Test animals

Details on test animals and environmental conditions:
- Source: Breeding farm VELAZ s.r.o., Koleč u Kladna, Czech Republic, RČH CZ 21760118
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 to 10 weeks
- Weight at study initiation: 18.20 – 21.71 g
- Housing: max 6 animals in macrolon cages with sterilized softwood shavings, monitored conditions, microbiologically defined background, according to internal SOP No.40
Cleaning and disinfection of animal room was regularly performed, as it is described in internal SOP No.10.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days
- Indication of any skin lesions: no clinical changes, all animals were examined during the acclimatisation period

- Temperature (°C): 22 ± 1 °C, permanently monitored
- Humidity (%): 41 – 58 %, permanently monitored
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle: 6am-6pm/6pm-6am
- Air changes (per hr): not specified

from: 29.09. 2010 (first day of administration after acclimatization)
to: 04.10. 2010 (necropsy)

Study design: in vivo (LLNA)

other: DAE 433 (mixture of 40% dimethylacetamide, 30% acetone and 30% ethanol)
The test substance was administered in the form of suspension in DAE 433.
Concentrations of test substance in application form:
20% (w/v) 200 mg/mL
2% (w/v) 20 mg /mL
0.2% (w/v) 2 mg /mL
No. of animals per dose:
Exposed groups – 15 females (5 animals in three groups)
Positive control group – 5 females
Negative control group – 5 females
Details on study design:
- Compound solubility:
The highest concentration 20% (w/v) was the technically practicable maximum concentration and was determined from the pilot experiment.
The appropriate suspensions of the test substance (20%, 2%, 0.2% w/v) was applied to three animals in volume 25 ul to the dorsum of each ear once a day morning for 3 consecutive days.
The suspensions were prepared before the start of application by mixing on magnetic stirrer and then were still mixed during application. The application was performed very slowly by micropipette to avoid losses caused by draining from the ear.
During the pilot experiment, slight effects were found in all three animals, and no macroscopic changes (after necropsy) were detected.

Animals were subjected to a clinical examination (health check) shortly after arrival. No clinical changes were recorded.
After acclimatization the animals have been randomly allocated to the dose groups (acc. to internal SOP No.42) and assigned animal numbers.

- Criteria used to consider a positive response:
Cell proliferation
Positive response: the stimulation index (SI) is ≥ 3, and the response increases in dose-related manner
Negative response: the stimulation index (SI) is < 3 without the dose – response relationship
Ambiguous response: the stimulation index is < 3, but the response increases in dose-related manner (dose–response relationship), and eventually statistical significance is observed.
Ear weight – irritation effect
If a statistically significant increase of ear weight together with clear concentration dependence of the effect is recorded, the inflammatory effect is considered as irritation induced by the test substance.

Positive result in cell proliferation reveals that the test substance could be a contact allergen. When positive irritation effect in animals is demonstrated simultaneously, the possibility can not be ruled out that the evaluation based on cell proliferation could be a false positive.

the same as in the pre-screen test (see above)

Note: no skin reactions and no significant clinical symptoms of intoxication throughout the experiment
Positive control substance(s):
other: Dinitrochlorobenzene (DNCB)
For statistical calculations the software Statgraphic ® Centurion (version XV, USA) was used. Statistical evaluation of measured parameters was performed at first by applying the non-parametric Kruskal-Wallis test for the comparison of the measured effect in all treatment groups with the vehicle control group, as global test, and then the non-parametric two-group Mann-Whitney rank test (probability level 0.05) for all two-group comparisons.

Results and discussion

Positive control results:
All animals in the positive control group showed these symptoms caused by the application of DNCB: decreased response to stimuli, well-defined erythema of skin (score 2) and clonospasm.

In vivo (LLNA)

Key result
< 3
Cellular proliferation data / Observations:
Examination of cell proliferation
The value of DPM and SI for positive control group was increased. In the positive control group, the SI was ≥ 3 (12.92) – the LLNA was efficient.
The SI for the test groups treated by the test substance at all three dose levels below the threshold.

Evaluation of irritating effect of the test substance
In the positive control group, the weight of ear target was statistically significantly increased as compared to the negative control group.
At all concentrations the weight of ears were relative well-balanced with the ear weight of the negative control group. Statistically significantly increase ear weight was not recorded.

Stimulation index (for incorporation of 3H-methyl thymidine) was calculated by dividing mean values from exposed groups and the positive control group by the corresponding mean value of the vehicle control group. The index for the vehicle control group was set at 1 by definition.

No animal died during the main experiment.
No symptoms of toxicity and local irritation were observed in animals of the negative control group and in all groups administered the test substance. All animals in the positive control group showed these symptoms caused by the application of DNCB: decreased response to stimuli, well-defined erythema of skin (score 2) and clonospasm.

Individual body weights before administration were relatively well balanced (result of random selection of animals into groups). Slightly decreased body weight before necropsy was recorded at the highest dose level. Body weight increments were calculated from values of day 6 before necropsy and day 1 before first application.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Under the given test conditions based on guidelines the animals exposed to the test substance, 2-(3',5'-Dichloro-2'-hydroxyphenyl)-4-quinazolinone, does not elicit sensitising response in LLNA assay.
Executive summary:

The test substance, 2-(3',5'-Dichloro-2'-hydroxyphenyl)-4-quinazolinone, was tested for the assessment of skin sensitisation potential with the murine local lymph node assay. This study is a part of the test substance health hazard evaluation.

The Local Lymph Node Assay (LLNA) with radionuclides was used. The testing was conducted according to the OECD Test Guideline No. 429, Skin sensitisation: Local Lymph Node Assay, Adopted 22nd July 2010.

In this study the contact allergenic potential of 2-(3',5'-Dichloro-2'-hydroxyphenyl)-4-quinazolinone, was evaluated after topical application to female BALB/c mice. Five mice per group were exposed on the dorsum of both ears once a day by test and control substances during 3 consecutive days. Primary proliferation of lymphocytes in the lymph node draining the site of application was evaluated by using of radioactive labelling. The ratio of the proliferation in treated groups to that in vehicle controls, termed the Stimulation Index (SI), was determined. Statistical evaluation of ear weight was performed for elimination of false positive findings with certain skin irritants.

Concentrations: positive control DNCB (dinitrochlorobenzene): 0.5% (w/v) and 2-(3',5'-Dichloro-2'-hydroxyphenyl)-4-quinazolinone: 20%, 2%, 0.2% (w/v) in vehicle (DAE 433).

The animals exposed to the test substance showed no pathological skin reactions and no other negative clinical symptoms of intoxication at all concentrations throughout the experiment.

The positive control substance DNCB elicited a reaction pattern with statistically significant increase in ear weight and Stimulation Index of cell proliferation reaching 12.92, which was in congruence with its expected mode of action as a contact allergen.

The test substance showed did not show a tendency to increased ear weight in any of concentrations tested. The result of skin irritation effect was considered as negative – it means the test substance did not cause irritation of skin.

The comparison of the Stimulation Indexes between treated groups and the control group revealed that the test substance did not cause increase in radioisotope incorporation into the DNA of dividing lymphocytes.

In conclusion, at the given experimental conditions the test substance 2-(3',5'-Dichloro-2'-hydroxyphenyl)-4-quinazolinone provided a negative result in LLNA test.