Phenol, mono- + distyrenated (MSP+DSP)

Skin sensitisation

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 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

1

Reference substance name:

Reaction mass of (1-phenylethyl)phenols and bis-(1-phenylethyl)phenols

Molecular formula:

not applicable

IUPAC Name:

Reaction mass of (1-phenylethyl)phenols and bis-(1-phenylethyl)phenols

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)

Results

Results 1

Parameter:

EC3

Value:

1.67

Results 2

Parameter:

SI

Value:

2.56

Test group / Remarks:

0.3% test substance in vehicle

Results 3

Parameter:

SI

Value:

3.43

Test group / Remarks:

3% test substance in vehicle

Results 4

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.