Registration Dossier

Administrative data

Description of key information

Skin Sensitisation: Negative for skin Sensitisation; OECD 442C; D Fleet. (2018).

Skin Sensitisation: Positive for skin Sensitisation; OECD 442D; F. Bailey. (2018).

Skin Sensitisation: Positive for skin Sensitisation; OECD 442E; M. Roth. (2018).

Skin Sensitisation: Positive for skin Sensitisation (Cat. 1B); OECD 429; E. Dony. (2018).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 Nov - 27 Nov 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
22 July 2010
Deviations:
yes
Remarks:
The relative humidity in the animal room was between approximately 30-65 % instead of 45–65% and the temperature was between 19 to 24°C instead of 20 to 24°C for several hours
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
06 July 2012.
Deviations:
yes
Remarks:
The relative humidity in the animal room was between approximately 30-65 % instead of 45–65% and the temperature was between 19 to 24°C instead of 20 to 24°C for several hours
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL

- Source and lot/batch No.of test material: Nalco/8247-22B
- Expiration date of the lot/batch: 31/ 10/2020
- Purity test date: not stated



RADIOLABELLING INFORMATION (if applicable): n/a
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL:
- Storage condition of test material: room temperature in the dark.
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble in dimethylformamide
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No
- Preliminary purification step (if any): No
- Final dilution of a dissolved solid, stock liquid or gel: No
- Final preparation of a solid: No

FORM AS APPLIED IN THE TEST (if different from that of starting material): n/a

OTHER SPECIFICS: No
Species:
mouse
Strain:
CBA
Remarks:
CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS B.V., Inc., Horst, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: Yes
- Microbiological status of animals, when known: Not reported
- Age at study initiation: 8 - 11 weeks
- Weight at study initiation: 17.1 - 22.3 g
- Housing: TMakrolon Type II (pre-test) / III (main study), with wire mesh top. Granulated soft wood bedding
- Diet (e.g. ad libitum): 2018C Teklad Global 18% protein rodent diet
(certified), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: At least 5 days
- Indication of any skin lesions: No

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25 ºC
- Humidity (%): 30 - 70 %
- Air changes (per hr): At least 15 changes/ hour
- Photoperiod (hrs dark / hrs light): 12 h: 12 h (light: dark)
- IN-LIFE DATES: Not reported
Vehicle:
dimethylformamide
Concentration:
10, 25, and 50%.
No. of animals per dose:
5
Details on study design:
:
PRE-SCREEN TESTS:
- Compound solubility: 50% in DMF
- Irritation: Yes 
- Systemic toxicity: Yes
- Ear thickness measurements: Yes
- Erythema scores: Yes

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response:
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.

TREATMENT PREPARATION AND ADMINISTRATION:
Each test group of 5 mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 10, 25, and 50% in DMF. The application volume, 25 µL/ear/day, was spread over the entire dorsal surface ( 8 mm) of each ear once daily for three consecutive days. Three further groups of mice (two vehicle control groups and one positive control group) were treated with an equivalent volume of the relevant vehicle alone or with the positive control item at 25% (w/v).
Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline containing 20 µCi of 3H-methyl thymidine (equivalent to 80.1 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.
Approximately five hours after treatment with 3HTdR all mice were euthanized by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death.
The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per animal). 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 approximately 18 hours for precipitation of macromolecules.
The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to scintillation vials with 10 mL of scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured in a -scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The -scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
All calculations conducted on the DPM values and the ear thickness were performed with a validated test script of “R”, a language and environment for statistical computing and graphics.Within the program a statistical analysis conducted on the DPM values and the ear thickness to assess whether the difference was statistically significant between the test item groups and negative control group. Statistical significance was set at the five per cent level (p < 0.05). Additionally, the Dean-Dixon-Test and Grubb’s Test were used for identification of possible outliers. An outlier was detected in both outlier tests (DPM value determined for animal 10). When this value was excluded from calculations, a mean S.I. value of 1.9 instead of 1.8 was obtained in the low dose group, and an EC3 value of 17.9% (w/v) was derived. Exclusion of this outlier value, however, did not change the overall test result.
Positive control results:
The animals treated with the positive control item alpha-Hexylcinnamaldehyde (25% (w/v)) showed a distinctly positive response (mean S.I. of 8.3), corroborating the validity of the assay.
Key result
Parameter:
SI
Value:
ca. 1.8
Test group / Remarks:
10%
Remarks on result:
other: Negative
Key result
Parameter:
SI
Value:
ca. 4
Test group / Remarks:
25%
Remarks on result:
other: Positive
Key result
Parameter:
SI
Value:
ca. 4.5
Test group / Remarks:
50%
Remarks on result:
other: positive
Key result
Parameter:
EC3
Value:
ca. 18.2
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA: 10 % and 25 % concentration.

DETAILS ON STIMULATION INDEX CALCULATION: Mean treatment group dpm / mean vehicle dpm

EC3 CALCULATION: The EC3 value calculated was 18.2 % (w/w).

CLINICAL OBSERVATIONS: No signs of systemic toxicity but some animal some animals showed an erythema of the ear skin with statistically significant and biologically relevant increase in ear thickness.


BODY WEIGHTS: Within normal limits

Table 2. Calculation of Stimulation Indices per Dose Group

Test item concentration

Group Calculation

Mean DPM per
animal (2 lymph nodes)a)

SD

S.I.

Vehicle Control Group (DMF)

891.8

222.5

1.0

10% α-phenyl-1H-benzimidazole-2-methanol

1597.2

277.3

1.8

25% α-phenyl-1H-benzimidazole-2-methanol

3568.2

1570.1

4.0*

50% α-phenyl-1H-benzimidazole-2-methanol

2387.2

1243.5

4.5*

Vehicle Control Group for the Positive Control Item (acetone/live oil (4+1, v/v))

1621.0

869.3

1.0

Positive Control Group
(25% α -HCA)

13502.6

3810.8

8.3*

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)

*  = Statistically significant (p<0.05) if compared to concurrent vehicle control

Table 3. Calculation of the EC3 value

Test Groups

Test item concentration %

S.I.

Group 2

10 (a)

1.8 (b)

Group 2

25 (c)

4.0 (d)

EC3 = (a-c) [(3-d)/(b-d)] + c = 18.2% (w/w)

a,b,c,d = Co-ordinates of the two pairs of data lying immediately above and below the S.I. value of 3 on the LLNA dose response plot.

Table 4. Ear Thickness in the Main Experiment

Test Group.

 

Ear thickness gain

Difference day 1 to 3

Difference day 1 to 6

Mean per dose group

Mean per dose group (%)

Mean per dose group

Mean per dose group (%)

1

1.5
±
4.9

0.7

6.5
±
4.5

2.9

2

4.5
±
9.9

2.0

10.0
±
7.3

4.2

3

0.0
±
4.7

0.0

2.5
±
10.9

1.1

4

8.5
±
6.3

3.7

11.0
±
5.2

4.6

5

3.5
±
12.6

1.5

4.5
±
3.3

1.9

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Based on the condition of the study, the test item,α-phenyl-1H-benzimidazole-2-methanol was found to be a skin sensitiser cat. 1B in accordance with the Globally Harmonized Classification System and to the Regulation (EC) No. 1272/2008.
Executive summary:

OECD 429 (2018): Three groups each of five female mice were treated once daily with the test item at concentrations of 10, 25, and 50% in DMF by topical application to the dorsum of each ear for three consecutive days. Three further groups each of five mice were treated with the vehicle for the test item (DMF) or the vehicle for the positive control item (acetone:olive oil (4+1 v/v)) only or with the positive control at 25%.

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 animal. 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 scintillation counter.

No mortality was reported and no signs of systemic toxicity were observed. Some animal some animals showed an erythema of the ear skin with statistically significant and biologically relevant increase in ear thickness.

Stimulation Indices of 1.8, 4.0, and 4.5 were determined with the test item at concentrations of 10, 25, and 50% (w/w) in DMF. A dose response was observed. The EC3 value calculated was 18.2 % (w/w).

The animals treated with the positive control item alpha-Hexylcinnamaldehyde (25% (w/v)) showed a distinctly positive response (mean S.I. of 8.3), corroborating the validity of the assay.

Based on the condition of the study, the test item,α-phenyl-1H-benzimidazole-2-methanol was found to be a skin sensitiser cat. 1B in accordance with the Globally Harmonized Classification System and to the Regulation (EC) No. 1272/2008.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

OECD 442C (2018): Solutions of α-phenyl-1h-benzimidazole-2-methanol  were successfully analysed by the validated DPRA- OECD 442C analytical method (Envigo analytical method FIA/M101/15) in both Cysteine and Lysine containing synthetic peptides. Solutions of the test item were successfully analysed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in the Cysteine containing synthetic peptide.  The result of 10.6% depletion places the test item in the reactivity class of “no to minimal” and therefore it is predicted by DPRA to be a non-skin sensitiser.   Under the conditions of this study, the test article was considered to be negative in the Direct Peptide Reactivity Assay according to EU CLP and UN GHS.

OECD 442D (2018): A single application of 12 concentrations of the test item was applied in cell culture medium (dilution factor of 2) with a final concentration of DMSO of 1%.  One well per plate was left empty (no cells). Cell seeding was conducted; (3 x 96-well plates for Luminescence; 2 x 96-well plate for MTT); 10,000 cells per well.  24 hours after seeding, the test  item and control items were applied and plates were incubated at 37oC, 5% CO2, ≥ 95% relative humidity for 48 ± 2 hours. Evaluation of luciferase activity by luminescence (3 plates) and cell viability by MTT testing (2 plates) was performed.  The test item induced statistically significant luciferase induction >1.5 in repetitions 1 and 2 but not in repetition 3. The EC1.5 values for repetitions 1 and 2 were calculated as 21.802 µM, and 15.494 µM, respectively. The maximum induction was observed at test concentrations of 250 µM for repetitions 1 and 2 and 62.5 µM for repetition 3. The IMAX values for repetitions 1, 2 and 3 were 2.368, 3.031 and 1.250, respectively. For reference, during test validation, sensitising proficiency chemicals produced IMAX values of up to 36-fold over untreated controls. Based on the condition of this study, the test item was classified as a Positive using the KeratinoSens prediction model in accordance to EU CLP and UN GHS.

OECD 442E (2018): The test item was prepared in DMSO which formed a stable suspension/dispersion or dissolved in DMSO at lower concentrations which was further diluted in culture medium when administered to THP-1 cells for 24 ± 0.5 hour. The test item was tested in 2 independent runs at concentration rage of 108, 130, 156, 187, 224, 269, 323 and 387 µg/mL. Precipitation was observed in four highest test item concentrations of both h-CLAT runs  and therefore were excluded from evaluation. The RFI of CD86 was equal or greater than 150% in at least one concentration of the first run and the RFI of CD86 and CD54 was equal or greater than 150% and 200%, respectively, in at least one concentration of the second independent run. All acceptance criteria are met and all controls are within the range of the historical control data. Based on the condition of the study, α-phenyl-1H-benzimidazole-2-methanol activated THP-1 cells under the test conditions of this study. Therefore the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

OECD 429 (2018): Three groups each of five female mice were treated once daily with the test item at concentrations of 10, 25, and 50% in DMF by topical application to the dorsum of each ear for three consecutive days. 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 animal. 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 scintillation counter. Stimulation Indices of 1.8, 4.0, and 4.5 were determined with the test item at concentrations of 10, 25, and 50% (w/w) in DMF. A dose response was observed. The EC3 value calculated was 18.2 % (w/w).

The animals treated with the positive control item alpha-Hexylcinnamaldehyde (25% (w/v)) showed a distinctly positive response (mean S.I. of 8.3), corroborating the validity of the assay.  Based on the condition of the study, the test item,α-phenyl-1H-benzimidazole-2-methanol was found to be a skin sensitiser cat. 1B in accordance with the Globally Harmonized Classification System and to the Regulation (EC) No. 1272/2008.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The test item,α-phenyl-1H-benzimidazole-2-methanol was found to be a skin sensitiser Cat. 1B in accordance with the Globally Harmonized Classification System and to the Regulation (EC) No. 1272/2008.