4,4'-Isopropylidenediphenol, ethoxylated

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

FROM 21 MAR 2012 to 25 JUN 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

July 22, 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

May 30, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-Isopropylidenediphenol, ethoxylated

EC Number:

500-082-2

EC Name:

4,4'-Isopropylidenediphenol, ethoxylated

Cas Number:

32492-61-8

Molecular formula:

C15H16O2.(C2H5O)n.(C2H5O)m sum of n+m: >1 - <4.5 moles EO

IUPAC Name:

2-[4-[2-[4-(2-hydroxyethoxy)phenyl]propan-2-yl]phenoxy]ethanol

Test material form:

liquid: viscous

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

Test System: Mice, CBA/CaOlaHsd
Rationale: Recognised at the recommended test system
Source: Harlan Laboratories B.V., The Netherlands
Identification: The animals were distributed into the test groups at random and identified by cage number. All animals belonging to the same experimental group were kept in one cage. In the main experiment, the animals were identified by tail tags. In the pre-experiment, animals were identified by cage number.
Acclimatisation: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.
Husbandry: The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.
Housing: Group
Cage Type: Makrolon Type II (pre-test), III (main study), with wire mesh top (EHRET GmbH, Germany)
Bedding: Granulated soft wood bedding (Rettenmaier & Sohne GmbH + Co, Germany)
Feed: Pelleted standard diet, ad libitum (Harlan Laboratories B.V, Germany)
Water: Tap water, ad libitum (Gemeindewerke, Germany)
Environment: Temperature: 22°C ± 2°C. Relative Humidity: 45-65% (acclimation period) and 45-65% (pre-test and main study). Artificial light 6.00 am - 6.00 pm. Air Changes: About 10/hour.
Age (beginning of treatment): Pre-test: 9-10 weeks. Main study: 8-9 weeks

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Concentration:

Test item concentrations of 10, 25 and 50% (w/w) were used.

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which could be technically used was a 50% (w/w) solution in methyl ethyl ketone.

To determine the highest non-irritant test concentration that does not induce signs of systemic toxicity at the same time, a pre-test was performed in two animals. Two mice were treated by topical application to the dorsal surface of each ear with test item concentrations of 25% (w/w) and 50% (w/w) once daily each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local irritation were documented and a score was used to grade a possible erythema of the ear skin. Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer (S0247 Kroeplin, 36381 Schlüchtern, Germany). Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Stiefel, Ø 8 mm corresponding to 0.5 cm2) and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if an erythema of the ear skin of a score value >/= 3 was observed at any observation time and/or if an increase in ear thickness of >/= 25% was recorded on day 3 or day 6. The measured ear weight was also considered in this evaluation. At the tested concentrations the animals did not show any signs of systemic toxicity. From day 2 to day 5, the animal treated with 50% test item concentration showed an erythema of the ear skin (Score 1). The animal treated with 25% test item concentration showed an erythema of the ear skin (Score 1) on day 2 and 4 (for detailed results of all observations and measurements, see Annex 1). On day 6, slight hair loss was observed in the neck region of both animals. Thus, the test item in the main study was assayed at 10, 25, and 50% (w/w).

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:
The proliferative response of the lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph nodes of each animal (DPM/animal) and as the ratio of 3HTdR incorporated into lymph node cells of lymph nodes of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.). Before DPM/animal values were determined, mean scintillation-background DPM was subtracted from test and control raw data.

A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
• First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, asindicated by the Stimulation Index.
• Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
- Test item preparation:
The test item was placed into an appropriate container on a tared balance and methyl ethyl ketone was added to achieve the required test item concentration. The different test item concentrations were prepared individually. The preparations were made freshly before each dosing occasion. Concentrations were in terms of material as supplied.

- Topical Application:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with test item concentrations of 10, 25, and 50% in methyl ethyl ketone. The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (approx. 8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone.

- Administration of 3H-Methyl Thymidine:
3 H-methyl thymidine (3 HTdR) was purchased from Hartmann Analytics, 38124 Braunschweig, Germany (specific activity, 2 Ci/mmol; concentration, 1 mCi/mL). Five days after the first topical application (day 6) 250 microL of phosphate-buffered saline (PBS) containing 20.0 microCi of 3 HTdR (equivalent to 79.9 microCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

The mean values and standard deviations were calculated in the body weight tables (as provided in the report), for the ear weights, the lymph node weights and lymph node cell count, and for the DPM values (group mean DPM ± standard deviation). A statistical analysis was conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between test item groups and the negative control group.
For all statistical calculations SigmaStat for Windows (Version 2.0) was used. A One-Way-analysis-of- Variance was used as statistical method. In case of significant results of the One-Way- ANOVA, multiple comparisons were performed with the Dunnett test. Statistical significance was set at the five per cent level (p < 0.05). The Dean-Dixon-Test and Grubb’s test were used for identification of possible outliers (performed with Microsoft Excel 2003). One outlier was identified.However, both biological and statistical significance were considered together.

Results and discussion

Positive control results:

The periodic positive control experiment was performed with α-hexyl cinnamaldehyde dissolved in acetone:olive oil(4:1 v/v) using CBA/CaOlaHsd mice. The results provided in the test report show that the positive control substance gave a Stimulation Index of between 3.73 and 10.77. This shows the positive control substance to be a skin sensitiser and confirms the sensitivity and reliability of the experimental technique.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

2.28

Test group / Remarks:

50%

Results 2

Parameter:

SI

Value:

1.58

Test group / Remarks:

25%

Results 3

Parameter:

SI

Value:

0.88

Test group / Remarks:

10%

Results 4

Parameter:

SI

Value:

1

Test group / Remarks:

Vehicle (methyl ethyl ketone)

Results 5

Parameter:

other: mean DPM desintegrations per minute (DPM)

Value:

1 101.1

Test group / Remarks:

50%

Remarks on result:

other: Mean DPM per animal (determined by dividing the sum of the measured values from lymph nodes of all animals within a group by the number of animals in that group (5 animals)

Results 6

Parameter:

other: mean DPM

Value:

762.5

Test group / Remarks:

25%

Results 7

Parameter:

other: mean DPM

Value:

425.5

Test group / Remarks:

10%

Results 8

Parameter:

other: mean DPM

Value:

482.9

Test group / Remarks:

Vehicle (methyl ethyl ketone)

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
The measured lymph node weights and –cell counts of all animals treated were recorded after sacrifice. A statistically significant increase in lymph node weights or –cell counts was not observed in comparison to the vehicle control group. However, the cut-off value for a positive response regarding the lymph node cell count index of 1.55 reported for BALB/c mice was exceeded in the high dose group (index of 1.57)

DETAILS ON STIMULATION INDEX CALCULATION
See 'Any other information on results incl. tables' section below

EC3 CALCULATION
The EC3 value of the test substance could not be calculated because all S. I.'s were below the threshold value of 3.

CLINICAL OBSERVATIONS:
No signs of systemic toxicity were observed during the study period. Signs of local skin irritation were also not observed.

BODY WEIGHTS
The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

SIGNS OF TOXICITY (including dermal irritation at the site of administration, if any, e.g. increased ear thickness).
No deaths occurred during the study period, and no signs of systemic toxicity were observed.
The measured ear weights of all animals treated were recorded after sacrifice. A statistically significant increase in ear weights was observed in all test item treated groups in comparison to the vehicle control group (p<0.05).
For BALB/c mice, a threshold for the ear weight index of 1.1 was reported for a positive response regarding ear skin irritation . At all tested concentrations, the index exceeded this threshold (indices of 1.2, 1.2, and 1.3). The increase of mean ear weight value ranged from 18.9% in the low dose group to 28.3% in the high dose group, thus indicating irritant properties of the test item.

Any other information on results incl. tables

Table 1: Calculation and results of individual data

Test Item Concentration (% w/w)	Group No.	Animal No.	DPM Values Measured	DPM-BG per animal (2 lymph nodes)^(a)	S. I.^(b)
-	-	BG I	18	-	-
-	-	BG II	19	-	-
0	1	1	201	183
		2	435	417
		3	287	269
		4^(c)	1267	1249
		5	317	299
10	2	6	230	212	0.4
		7	611	593	1.2
		8	310	292	0.6
		9	682	664	1.4
		10	387	369	0.8
25	3	11	441	423	0.9
		12	483	465	1.0
		13	746	728	1.5
		14	966	948	2.0
		15	1269	1251	2.6
50	4	16	917	899	1.9
		17	1266	1248	2.6
		18	1484	1466	3.0
		19	1359	1341	2.8
		20	572	554	1.1

 

BG = Background (1 mL 5% trichloroacetic acid) in duplicate

S.I. = Stimulation Index (values of the test item groups related to the mean value of the control group)

^(a) = Values corrected for mean background levels (BG I and BG II)

^(b) = Stimulation Indices relative to the mean of the control group (Group 1, animal no. 1-5)

^(c) = The value for animal 4 was identified as an outlier but was not excluded from the calculation, since the value is well within the range of the historical vehicle control data

 

Table 2: Calculation of Stimulation Indices per dose group

Test item concentration	Group Calculation
	Mean DPM per animal^(a); (c)	SD^(b)	S.I.
Vehicle (methyl ethyl ketone)	482.9	436.1	1.00
10% Pluriol BP 40E	425.5	194.6	0.88
25% Pluriol BP 40E	762.5	345.7	1.58
50% Pluriol BP 40E	1101.1*	371.6	2.28

^(a) Mean DPM/animal was determined by dividing the sum of the measured values from lymph nodes of all animals within a group by the number of animals in that group (5 animals)

^(b) SD = Standard Deviation

^(c) The DPM value for animal 4 was identified as an outlier but not excluded from the calculation

* Statistically significant increase vs. control group (p <0.05)

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this study Stimulation Indices (S.I.) of 0.88, 1.58 and 2.28 were determined with the test item at concentrations of 10, 25 and 50% (w/w), respectively. A test item is regarded as a sensitiser in the LLNA if exposure to one or more test item concentrations results in an S.I. of 3 or more. Therefore, the test item is not considered to be a skin sensitiser under the test conditions of this study.

Executive summary:

In a dermal sensitisation study (according to OECD TG 429), the skin sensitisation of the test substance (purity: 99.2%) in methyl ethyl ketone was measured using the Local Lymph Node Assay (LLNA) in young adult female CBA/CaOlaHsd mice (n= 5/group). The LLNA was performed using test item concentrations of 10, 25 and 50% (w/w). The highest concentration tested was the highest concentration that could be technically used and applied whilst avoiding systemic toxicity and excessive local skin irritation (as determined by a pre-experiment). The animals did not show any signs of systemic toxicity or local skin irritation during the course of the study, and no cases of mortality were observed. In this study Stimulation Indices (S.I.) of 0.88, 1.58, and 2.28 were determined with the test item at concentrations of 10, 25, and 50% (w/w), respectively. A test item is regarded as a sensitiser in the LLNA if exposure to one or more test item concentrations results in an S.I. of 3 or more. Therefore, the test item is not considered to be a skin sensitiser under the test conditions of this study.