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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 April - 1 May 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted: 2001
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

1
Chemical structure
Reference substance name:
6-amino-5-chloro-2-cyclopropylpyrimidine-4-carboxylic acid
EC Number:
617-769-9
Cas Number:
858956-08-8
Molecular formula:
C8H8ClN3O2
IUPAC Name:
6-amino-5-chloro-2-cyclopropylpyrimidine-4-carboxylic acid

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/JHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague Dawley, Frederick, Maryland, USA
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 9 weeks
- Weight at study initiation: 21.3 - 23.2 g
- Housing: 2 animals housed in stainless steel wire-mesh cages suspended above cage boards during quarantine, individual housed in stainless steel wire-mesh cages suspended above cage boards during dosing and resting phases of the study
- Diet: PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002, ad libitum
- Water: ad libitum
- Acclimation period: at least 6 days
- Indication of any skin lesions: There were no indications of any ear abnormalities (e.g., torn, scratched), clinical signs of disease or injury.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 26
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
1, 5, 25 and 50%
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: The test substance was prepared in vehicle. Due to insolubility of the test substance, doses greater than 50% were not evaluated.
- Systemic toxicity: Animals were observed for changes in body weight and weight gain and any gross signs of disease or injury.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: ³H-thymidine incorporation determined by beta counter
- Criteria used to consider a positive response: A stimulation index (SI) was derived for each experimental group by dividing the mean dpm of each experimental group by the mean dpm of the vehicle control group. A response is considered positive when the SI is ≥ 3.0 together with consideration of dose response and, where appropriate, statistical significance.

TREATMENT PREPARATION AND ADMINISTRATION: 25 µL of test substance preparations, vehicle or positive control were administered topically to the dorsum of each mouse ear for 3 consecutive days (test days 0-2). Test days 3-4 were days of rest followed by intravenous injection of 20 μCi of ³H-Thymidine per mouse on test day 5. Approximately 5 h after the injection, animals were sacrificed by carbon dioxide asphyxiation, draining auricular lymph nodes were removed, and single cell suspensions were prepared. The single cell suspensions were incubated at 2-8 °C overnight. On test day 6, the single cell suspensions were counted on a beta counter. The counts per minute (cpm) data were converted to disintegrations per minute (dpm).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Body weight and body weight gain:
Preliminary test: Levene’s test for homogeneity and Shapiro-Wilk test for normality
If preliminary test is not significant: One-way analysis of variance followed by Dunnett's test
If preliminary test is significant: Kruskal-Wallis test followed by Dunn's test

Lymph node dpm data:
Preliminary test: Test for lack of trend or Levene’s test for homogeneity and Shapiro-Wilk test for normality
If preliminary test is not significant: Sequential application of the Jonckheere-Terpstra trend test or One-way analysis of variance followed by Dunnett's test
If preliminary test is significant: Preliminary tests for pairwise comparison or Kruskal-Wallis test followed by Dunn's test

Significance was judged at p < 0.05 except for dpm data that were judged at p < 0.01. Lymph node dpm data were transformed to Log to obtain normality or homogenous variances.

When possible, an EC3 value for the SI data was derived from linear interpolation of points on the dose-response curve immediately above and below the 3-fold threshold. The equation used for calculation of EC3 was:
EC3 = c + [(3 - d)/(b - d)] × (a - c)
where:
a = the lowest concentration giving stimulation greater than 3
b = the actual SI caused by a
c = the highest concentration failing to produce an SI of 3
d = the actual SI caused by c

Results and discussion

Positive control results:
A 25% concentration of the positive control produced a SI of 13.28 and thus a dermal sensitization response in mice.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.8
Test group / Remarks:
1%
Key result
Parameter:
SI
Value:
1.69
Test group / Remarks:
5%
Key result
Parameter:
SI
Value:
2.29
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
1.62
Test group / Remarks:
50%
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
No statistically significant increases in cell proliferation measurements compared to the vehicle control group were observed at any test concentration. Stimulation indices of less than 3.0 were observed at all test concentrations.

DETAILS ON STIMULATION INDEX CALCULATION
A stimulation index (SI) was derived for each experimental group by dividing the mean dpm of each experimental group by the mean dpm of the vehicle control group.

EC3 CALCULATION
Not calcuable for this study.

CLINICAL OBSERVATIONS
Dehydration was observed in one animal in the positive control group on test day 2. This mouse was found dead on test day 3. Gross necropsy revealed no abnormalities.

BODY WEIGHTS
No statistically significant differences in mean body weights and body weight gains compared to the vehicle control group were observed at any test concentration.

Any other information on results incl. tables

 Table 1: Results of the Stimulation Index data

Group

Test material

number of animals

Individual (dpm)

Stimulation Index

1

Vehicle control

5

414.25

186.25

456.25

1658.25

299.25

 

Mean ± Standard deviation (dpm)

602.85 ± 599.30

-

2

Test substance 1%

5

1585.25

632.25

1070.25

1368.25

765.25

 

Mean ± Standard deviation (dpm)

1084.25 ± 399.35

1.80

3

Test substance 5%

5

1702.25

899.25

485.25

1332.25

684.25

 

Mean ± Standard deviation (dpm)

1020.65 ± 494.02

1.69

4

Test substance 25%

5

2341.25

695.25

667.25

1730.25

1481.25

 

Mean ± Standard deviation (dpm)

1383.05 ± 713.05

2.29

5

Test substance 50%

5

1666.25

852.25

499.25

526.25

1342.25

 

Mean ± Standard deviation (dpm)

977.25 ± 513.68

1.62

6

 

Positive control 25%*

4**

6195.25

**

9308.25

7374.25

9136.25

 

Mean ± Standard deviation (dpm)

8003.50 ± 1488.98

13.28

* Data were not included in the statistical analysis of the test substance groups

** One mouse was found dead on test day 3

dpm: disintegrations per minute

 

 Table 2: Mean body weight and body weight gains of female mice

 

Mean body weight (g) ± Standard deviation

Day

Group 1

Group 2

Group 3

Group 4

Group 5

Group 6

 

vehicle control

Test substance 5%

Test substance 25%

Test substance 50%

Test substance 1%

Positive control 25%

0

22.2 ± 0.6

22.3 ± 0.6

22.3 ± 0.7

22.4 ± 0.6

22.0 ± 0.5

22.4 ± 0.8

5

22.3 ± 0.9

22.3 ± 0.8

22.0 ± 0.5

21.8 ± 1.2

21.4 ± 0.5

22.0 ± 0.7

 

Mean body weight gains (g)

0-5

0.1 ± 0.8

-0.0 ± 0.4

-0.3 ± 0.8

-0.6 ± 0.8

-0.6 ± 0.8

-0.6 ± 1.3

 

 

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008
Conclusions:
Under the conditions of the conducted LLNA test, the test substance did not exhibit sensitising properties.
CLP: not classified