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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Test material form:
liquid
Details on test material:
- additional information as appropriate is presented in the respective study record
Specific details on test material used for the study:
- Name of test material (as cited in study report): Novares LS 500; phenol, styrenated
- Source and lot/batch No.of test material: RÜTGERS Novares GmbH, batch No. 28324
- Composition of test material: composition is specified in IUCLID Sect. 13 - Assessment reports under Certificate of Analysis_Novares LS 500_phenol, styrenated
- Substance type: organic
- Purity test date: 30.11.2012
- Stability under test conditions: no measured data; based on chemical structure assumed to be stable

In vivo test system

Test animals

Species:
mouse
Strain:
Balb/c
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Breeding farm VELAZ s.r.o., Koleč u Kladna, Czech Republic
- Age at study initiation: 8 - 10 wks
- Weight at study initiation: 17 - 19 g
- Housing: macrolon cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 3
- Humidity (%): 30 – 70
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:
other: DAE 433
Concentration:
mixture of 40% dimethylacetamide, 30% acetone and 30% ethanol
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: dispersion
- Irritation: no
- Lymph node proliferation response: not examined


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:
Stimulation index (SI) is ≥ 3
Response increases in dose-related manner
Criteria for irritation potential: individual and mean ear weight


TREATMENT PREPARATION AND ADMINISTRATION:
Days 1-3: 25 µL test suspension in DAE 433 , 1x on day 1, 2, and 3
Days 4 and 5: no treatment
Day 6: Injection of 250 μL of phosphate-buffered saline (PBS) containing 7.5 x105 Bq (c. 21 µCi) of 3H-methyl thymidine
into all test and control mice via the tail vein. Five hours later, the animals were killed.



Positive control substance(s):
other: DNCB (dinitrochlorbenzene, 0.5% (w/v) solution)
Statistics:
Non-parametric Kruskal-Wallis test, and then the non-parametric two-group Mann-Whitney rank test (probability level 0.05) was applied to all two-group comparisons [using software Statgraphic ® Centurion (version XV, USA)].

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
EC3
Value:
1.67
Parameter:
SI
Value:
2.56
Test group / Remarks:
0.3% test substance in vehicle
Parameter:
SI
Value:
3.43
Test group / Remarks:
3% test substance in vehicle
Parameter:
SI
Value:
27.07
Test group / Remarks:
30% test substance in vehicle
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Proliferation data were determined as mean (radioactivity counts - DPM) of five test animals per group

DETAILS ON STIMULATION INDEX CALCULATION
SI was calculated using the following equation: SI = mean of test groups (DPM) / mean of control group (DPM)

EC3 CALCULATION
Linear interpolation with EC3 = c + [(3–d)/(b–d)]×(a–c), where the data points lying immediately above and below the SI value of 3 on the LLNA dose–response plot having the coordinates (a,b) and (c,d), respectively.
(a = dose concentration immediately above SI=3, b = SI immediately above 3)
(c = dose concentration immediately below SI=3, d = SI immediately below 3)

Any other information on results incl. tables

Table 1: Individual and mean radioactivity in cell suspensions (Report Table 4)

Animal No.

Activity (DPM)

NC

(1-5)

PC

(6-10)

30%

(11-15)

3%

(16-20)

0.3%

(21-25)

1

550.98

11374.69

13868.92

1764.06

1504.40

2

485.39

10401.94

15745.06

1472.17

1129.52

3

568.93

10347.82

13338.82

1552.57

1218.14

4

526.30

8837.38

15573.65

1811.12

1337.00

5

596.74

9493.34

15327.01

2756.16

1804.05

 Group

mean

545.67

10091.03

14770.69

1873.02

1398.62

SI

1.00

18.49

27.07

3.43

2.56

NC = vehicle control; PC = positive control

Table 2: Individual and average ear weights (Report Table 5)

Animal No.

Weight of ear biopsies (milligrams)

NC

(1-6)

PC

(7-12)

30%

(13-18)

3%

(19-24)

0.3%

(25-30)

1

24.5

27.4

30.6

21.4

22.0

2

25.2

27.5

26.7

21.3

21.2

3

23.8

26.5

35.2

22.4

18.8

 4

23.4

26.7

30.6

22.9

20.2

5

24.7

27.2

30.2

21.8

22.3

 Group mean

24.32

27.06*

30.66*

21.96*

20.90*

SD

0.72

0.44

3.02

0.68

1.43

NC = vehicle control; PC = positive control

* statistically significant with p =< 0.05, Mann-Whitney test

Table 3:  Summary table (Report Table 6)

Group

Radioisotope incorporation

in lymph nodes

Ear weight

Mean DPM

SI

Mean (mg)

NC

545.67

1.0

24.32

PC

10091.03

18.49+

27.06

30%

14770.69

27.07+

30.66

3%

1873.02

3.43+

21.96

0.3%

1398.62

2.56

20.90

Bold figures with + = values >= 3

Applicant's summary and conclusion

Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
In a LLNA according to OECD TG 429, an EC3 of 1.67 was determined. This requires phenol, styrenated (LS 500) to be classified as skin sensitising Cat. 1A.