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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:
07 Sep - 11 Oct 2017
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted: July 22, 2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
30 May 2008
GLP compliance:
yes (incl. certificate)
Remarks:
GLP-Landesleitstelle Bayern, Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo (formed partially from Harlan in September 2015), 5800 AN Venray, The Netherlands
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF
- Age at study initiation: 8-9 weeks
- Housing: the animals were kept in groups of 5 animals in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding
- Diet: Altromin 1324 maintenance diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least five days
- Indication of any skin lesions: none

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%):55 ± 10
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0.5, 1 and 2 g/mL (suspension)
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS: two animals per dose were treated by topical application with the test item on three consecutive days at concentrations of 1 and 2 g/mL in acetone/olive oil 4:1 (AOO) to the entire dorsal surface of each ear. One further animal was treated with AOO (neat) and served as negative control.
- Compound solubility: the vehicle was chosen as it was suitable at the highest concentration.
- Irritation: the mice were observed daily for local skin irritation to the application site.
- Systemic toxicity: the mice were observed daily for any clinical signs of systemic toxicity, and body weights were recorded pre-test and prior to termination.
- Ear thickness measurements: performed on day 1 (pre-dose), day 3 (approximately 48 hours after the first dose) and day 6.
- Erythema scores: No erythema (0), very slight erythema (1), well-defined erythema (2), moderate to severe erythema (3), severe erythema to eschar formation preventing grading of erythema (4).

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine (3HTdR) incorporation determined by β-scintillation counting
- Criteria used to consider a positive response: A substance is regarded as a “sensitizer” in the LLNA if at least one concentration of the test item results in a 3-fold or greater increase in 3HTdR incorporation into lymph node cells of the test group animals, relative to that recorded for the lymph nodes of control group animals. Any test item failing to produce a 3-fold or greater increase in 3HTdR incorporation is classified as a "non-sensitizer".
If the results allowed the EC3 value could also be calculated. The EC3 value is the concentration of test item expected to cause a 3-fold increase in 3HTdR incorporation. The equation used for the calculation of EC3 is:
EC3 = c + [[(3-d)/(b-d)] x (a-c)]
a = lowest concentration giving stimulation index >3
b = actual stimulation index caused by 'a'
c = highest concentration failing to produce a stimulation index of 3
d = actual stimulation index caused by 'c'

- Other: The animals were observed for signs of toxicity prior to the application and once daily after treatment. The body weight was recorded prior to the application and at the end of the test period (prior to the treatment with 3HTdR).

TREATMENT PREPARATION AND ADMINISTRATION: 25 μL of the test compound in concentrations of 0.5, 1 and 2 g/mL in AOO and negative control (AOO) was applied to the dorsal surface of each ear of each mouse on Day 1, 2 and 3. On Day 6 an injection of 250 μL phosphate buffered saline (PBS) containing 20 μCi of 3H-methyl thymidine (3H-TdR) was made into the tail vein of each experimental mouse. Five hours later, the draining auricular lymph node of each ear was excised into PBS. A single cell suspension of pooled lymph node cells was prepared per experimental group by gentle mechanical disaggregation through a 200-mesh stainless steel gauze and rinsed with PBS. The precipitates were incubated for approximately 18 h at approximately 4 °C, centrifuged, resuspended in 1 mL 5% TCA (trichloroacetic acid) and transferred to 7 mL scintillation fluid before β-scintillation counting.
Positive control substance(s):
other: Phenylene- diamine

Results and discussion

Positive control results:
A positive control was performed concomitantly using 5 animals, the result showed the positive-control substance exceeded the stimulation index of 3 confirming the reliability of the test system.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.4
Test group / Remarks:
0.5 g/mL
Key result
Parameter:
SI
Value:
2.9
Test group / Remarks:
1 g/mL
Key result
Parameter:
SI
Value:
1.7
Test group / Remarks:
2 g/mL
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION
The SI of the 0.5, 1 and 2 mg/mL treatment group was 1.4, 2.9 and 1.7, respectively.

EC3 CALCULATION
The EC3 value could not be calculated as the stimulation indices of all concentrations were below 3.

CLINICAL OBSERVATIONS:
All animals survived throughout the test period without showing any clinical signs.

BODY WEIGHTS
All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the study.

Any other information on results incl. tables

Table 1: Radioactive Determination of the Test Substance Groups – Main Study

POS

CPM

Test Item

Conc. [g/mL]

Animal number

DPM

DPM-mean background

DPM/ Node

Stimulation Index

86

87

88

89

90

MV

SD

614.0

512.0

450.0

605.0

992.0*

545.3

68.0

Negative Control (AOO)

neat

101

102

103

104

105

MV

SD

1229.0

1032.0

901.0

1210.0

1997.0*

1093.0

134.9

1192.2

995.2

864.2

1173.2

n.d.

1056.2

134.9

596.1

497.6

432.1

586.6

n.d.

528.1

67.4

1.0

71

72

73

74

75

MV

SD

1228.0

802.0

1231.0

944.0

456.0

932.2

290.0

Frits in AOO

2

1

2

3

4

5

MV

SD

2494.0

1610.0

2477.0

1894.0

912.0

1877.4

590.5

2457.2

1573.2

2440.2

1857.2

875.2

1840.6

590.5

1228.6

786.6

1220.1

928.6

437.6

920.3

295.3

2.3

1.5

2.3

1.8

0.8

1.7

0.6

76

77

78

79

80

MV

SD

948.0

1593.0

1459.0

2202.0

1420.0

1524.4

402.8

Frits in AOO

1

6

7

8

9

10

MV

SD

1911.0

3202.0

2933.0

4425.0

2844.0

3063.0

808.2

1874.2

3165.2

2896.2

4388.2

2807.2

3026.2

808.2

937.1

1582.6

1448.1

2194.1

1403.6

1513.1

404.1

1.8

3.0

2.7

4.2

2.7

2.9

0.8

81

82

83

84

85

MV

SD

1154.0

657.0

1602.0

170.0

223.0

761.2

549.8

Frits in AOO

0.5

11

12

13

14

15

MV

SD

2329.0

1320.0

3211.0

342.0

446.0

1529.6

1104.1

2292.2

1283.2

3174.2

305.2

409.2

1492.8

1104.1

1146.1

641.6

1587.1

152.6

204.6

746.4

552.0

2.2

1.2

3.0

0.3

0.4

1.4

1.0

96

97

98

99

100

MV

SD

6.0

14.0

7.0

55.0

10.0

18.4

18.5

Background

Szinti and TCA

 

 

 

 

 

 

MV

SD

11.0

28.0

15.0

110.0

20.0

36.8

37.0

 

 

 

 

 

0.0

 

 

 

 

 

 

0.0

 

 

 

 

 

 

0.0

 

* = outlier, failed Grubbs, Nalimov and Dixon; n.d. = not determined

If not noted individually, the results include both lymph nodes of an animal.

POS = position in counter; CPM = counts per minute; Conc. = concentration; DPM = disintegrations per minute; SD = standard deviation; MV = mean value; Szinti = scintillation fluid; TCA = trichloroacetic acid

Applicant's summary and conclusion

Interpretation of results:
other: evaluation according to CLP/EU GHS criteria currently not possible