Phenolphthalein

Endpoint summary

Description of key information

In a study performed according OECD TG 429 the test item is not a skin sensitizer (reference 7.4.1 -1).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 03 , 2010 - August 18, 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2002

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V. Postbus 6174, 5960 AD Horst / The Netherlands
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 22 +/- 1.5 g
- Housing: single caging, Makrolon Type II
- Diet: pelleted standard diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22± 2
- Humidity (%): 45-95
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

dimethylformamide

Concentration:

5, 10, 25%

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which can be technically used was a 25 % solution in dimethylformamide. Vortexing and warming to 37°C were used to formulate the test item.
- Irritation: To determine the highest non-irritant test concentration that did not induce signs of systemic toxicity at the same time, a pre-test was performed in two animals. Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with concentrations of 10 and 25% once daily each on three consecutive days. Furthermore, prior to the first application of the test item and before sacrifice the body weight was determined.
- Systemic toxicity: In the pre-test clinical signs were recorded within 1 hour and 24 ± 4 hours after each application as well as on day 7. At the tested concentrations the animals did not show any signs of irritation or systemic toxicity.

MAIN STUDY
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear (left and right) with different test item concentrations of 5, 10, and 25% (w/w) in dimethylformamide. The application volume, 25 µL, was spread over the entire dorsal surface of each ear once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).

3H-methyl thymidine (3HTdR) was purchased from Hartmann Analytic, 38124 Braunschweig, Germany (specific activity, 2 Ci/mmol; concentration, 1 mCi/mL). Five days after the first topical application, all mice were administered with 250 µl of 81.9 uCi/mL 3HTdR (corresponds to 20.5 uCi 3HTdR per mouse) by intravenous injection via a tail vein.

Prior to the first application of the test item and prior to treatment with 3HTdR the ear thickness was determined using a micrometer (S0247 Kroeplin, 36381 Schlüchtern, Germany).

Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital-Natrium (Release®, WDT, D-30827 Garbsen, Germany). The draining lymph nodes were rapidly excised and pooled per group (8 nodes per group). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to plastic scintillation vials with 10 mL of 'Ultima Gold' scintillation liquid (Perkin Elmer (LAS) GmbH, 63110 Rodgau, Germany) and thoroughly mixed. The level of 3HTdR incorporation was then measured on a ß-scintillation counter (Tricarb 2900 TR, Perkin Elmer (LAS) GmbH, 63110 Rodgau, Germany). Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The ß-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

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, as indicated 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.

In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:

Mortality / Viability: once daily (week day) from experimental start to necropsy.
Body weights: In the pre-test: prior to the first application and prior to sacrifice. In the main experiment: prior to the first application and prior to treatment with 3HTdR.
Clinical signs (local / systemic): In the pre-test clinical signs were recorded within 1 hour and 24 ± 4 hours after each application as well as on day 7. In the main experiment clinical signs were recorded within 1 hour after each application, and 24 ± 4 hours after the first and second application as well as on the day of preparation. Especially the treatment sites were observed carefully.

Positive control substance(s):

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

Statistics:

The ANOVA (Dunnett-test) was conducted on the values determined for the ear thickness to assess whether the difference is statistically significant between test item groups and negative control (vehicle) group. However, both biological and statistical significance were considered together.

Positive control results:

a-Hexylcinnamaldehyde was positive for skin sensitisation (SI of 3.41 at 10% test concentration and SI of 6.14 at 25% test concentration)

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

control group

Key result

Parameter:

SI

Value:

1.06

Test group / Remarks:

5% test item

Key result

Parameter:

SI

Value:

1.55

Test group / Remarks:

10% test item

Key result

Parameter:

SI

Value:

1.24

Test group / Remarks:

25% test item

Key result

Parameter:

SI

Value:

2.04

Test group / Remarks:

5% positive control

Key result

Parameter:

SI

Value:

3.41

Test group / Remarks:

10% positive control

Key result

Parameter:

SI

Value:

6.14

Test group / Remarks:

25% positive control

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (stimulation index). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.

EC3 CALCULATION : not performed

CLINICAL OBSERVATIONS: No deaths occurred during the study period. No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

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.

The measured ear thickness of all animals treated was recorded prior to the 1st application and prior to treatment with 3HTdR. A statistically relevant increase in ear thickness was not observed.

Table 1: Results

Test item concentration % (w/w)	Group	Measurement DPM	Calculation			Result
			DPM-BG a)	number of lymph nodes	DPM per lymph node b)	S.I.
—	BGI	78	—	—	—	—
—	BG II	58	—	—	—	—
—	1	2962	2894	8	361.8	1.00
5	2	3150	3082	8	385.3	1.06
10	3	4555	4487	8	560.9	1.55
25	4	3647	3579	8	447.4	1.24

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

1= Control Group

2-4= Test Group

S.I.= Stimulation Index

a)= The mean value was taken from the figures BG I and BG II

b)= Since the lymph nodes of the animals of a dose group were pooled, DPM/node was
determined by dividing the measured value by the number of lymph nodes pooled

EC3 value could not be calculated, since all S.I's are below 3.

Table 2: Test results with positive control

Experiment performed in May 2010.

Positive control substance: a-Hexylcinnamaldehyde

Vehicle: acetone:olive oil (4+1)

Test item concentration % (w/v)	Group	Measurement DPM	Calculation			Result
			DPM-BG a)	number of lymph nodes	DPM per lymph node b)	S.I.
—	BG I	13	—	—	—	—
—	BG II	12	—	—	—	—
0	1	4561	4549	8	568.6	1.00
5	2	9275	9263	8	1157.8	2.04
10	3	15507	15495	8	1936.8	3.41
25	4	27961	27949	8	3493.6	6.14

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

1 = Control Group

2-4 = Test Group

S.I. = Stimulation Index

a)= The mean value was taken from the figures BG I and BG II

b)= Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

 

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is not a skin sensitiser in the LLNA.

Executive summary:

A study according OECD TG 429 was conducted to investigate a possible contact allergenic potential of the test item, three groups each of four female mice were treated once daily with the test item at concentrations of 5, 10, and 25% (w/w) in dimethylformamide by topical application to the dorsum of each ear (left and right) for three consecutive days. A control group of four mice was treated with the vehicle (dimethylformamide) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a p-scintillation counter. All treated animals survived the scheduled study period and no signs of systematic toxicity or local irritation were observed. A statistically relevant increase in ear thickness was not observed. A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.

In this study Stimulation Indices of 1.06, 1.55, and 1.24 were determined with the test item at concentrations of 5, 10, and 25% in dimethylformamide. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than 3. The test item wasnot a skin sensitiser in this assay.

Reference 2

Endpoint:

skin sensitisation: in vitro

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

A study according OECD TG 429 was conducted to investigate a possible contact allergenic potential of the test item, three groups each of four female mice were treated once daily with the test item at concentrations of 5, 10, and 25 % (w/w) in dimethylformamide by topical application to the dorsum of each ear (left and right) for three consecutive days. A control group of four mice was treated with the vehicle (dimethylformamide) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a p-scintillation counter.All treated animals survived the scheduled study period and no signs of systematic toxicity or local irritation were observed. A statistically relevant increase in ear thickness was not observed.A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.

In this study Stimulation Indices of 1.06, 1.55, and 1.24 were determined with the test item at concentrations of 5, 10, and 25 % in dimethylformamide. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than 3.The test item was not a skin sensitiser in this assay.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on skin sensitisation, the test item does not require classification according to Regulation (EC) No 1272/2008 (CLP), as amended for the fourteenth time in Regulation (EU) 2020/217.