[No public or meaningful name is available]

Endpoint summary

Description of key information

DPRA (OECDTG442C): negative

KeratinoSensTM assay (OECDTG442D): inconclusive

LLNA (OECDTG429): not sensitising

 

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:

04 January 2021 - 27 January 2021

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:

(July 2010)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EC No 640/2012, Part B: Skin sensitization: "Local Lymph Node Assay"

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

(March 2003)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately 10 weeks old
- Weight at study initiation: 20.8 to 25.2 g
- Housing: Animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon MIII type; height 18 cm.) containing sterilized wooden fibers as bedding material.
- Diet: free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: free access to municipal tap-water.
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 40 to 44
- Air changes (per hr): ten or greater
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 06 January 2021 To: 25 January 2021

Vehicle:

dimethylformamide

Concentration:

0, 10, 25 and 40% (w/w)

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TEST:
Two test item concentrations were tested; a 25% and 40% concentration. The highest concentration was the highest concentration that could be prepared homogeneously.
The test system, procedures and techniques were identical to those used in the main study except that the assessment of lymph node proliferation and necropsy were not performed.
Two young adult females per concentration were selected. Each animal was treated with one
concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 14 cm). Ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on Days 1 and 3, and on Day 6.
Animals were sacrificed after the final observation.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group. If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer, based on the test guideline.

ANIMAL ASSIGNMENT
Three groups of five animals were treated with one test substance concentration per group. One group of five animals was treated with vehicle.

TREATMENT PREPARATION AND ADMINISTRATION:
Test substance preparation: Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. Amber glassware was used for preparation of formulations.
The dosing formulations were prepared daily and dosed within 4 hours after adding the vehicle to the test item. The dosing formulations were kept at room temperature until dosing. The dosing formulations were stirred until and during dosing.

Rationale for vehicle: The vehicle was chosen from the vehicles specified in the test guideline (in order of preference): Acetone/Olive oil (4:1 v/v), N,N-dimethylformamide, methylethylketone,
propylene glycol, dimethylsulfoxide and 1% Pluronic© L92 in Elix water (in case an aqueous vehicle is suitable). The vehicle was selected on the basis of maximizing the solubility based on trial preparations performed at Charles River Den Bosch and on information provided by the Sponsor. Trial preparations were performed to select the suitable vehicle and to establish a suitable formulation procedure. These trials were not performed as part of this study and these
preparations were not used for dosing. Raw Data of these trials were retained by the Test Facility. There was no information available about the stability and solubility of the test item in vehicle.

Induction - Days 1, 2 and 3; Excision of nodes - Day 6; Tissue processing for radioacitivity - Day 6; Radioactivity measurements - Day 7; Performed according to test guidelines.

Observations:
Mortality/Moribundity: Twice daily.
Body weights: On Day 1 (pre-dose) and Day 6 (prior to necropsy).
Clinical signs: Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
Irritation: Once daily on Days 1-6 (on Days 1 - 3 within 1 hour after dosing) according to a numerical scoring system (see table 1 below). Furthermore, a description of all other (local) effects was recorded according to guidelines.

Necropsy: No necropsy was performed, since all animals survived until the end of the observation period.

Positive control substance(s):

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

Statistics:

Not performed.

Positive control results:

The six-month reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.

Key result

Parameter:

SI

Value:

2.8

Test group / Remarks:

10%

Key result

Parameter:

SI

Value:

2.2

Test group / Remarks:

25%

Key result

Parameter:

SI

Value:

2.7

Test group / Remarks:

40%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA:
Mean DPM/animal values: concentration, 10, 25 and 40% were, 641, 501 and 614 DPM, respectively. Mean DPM/animal values vehicle control: 229 DPM.

CLINICAL OBSERVATIONS:
No mortality occurred and no clinical signs of systemic toxicity were observed in the animals.

BODY WEIGHTS:
Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

SIGNS OF TOXICITY:
The very slight irritation of the ears as shown by all animals treated at 25% and 40% between Days 1 and 3 was considered not to have a toxicologically significant effect on the activity of the nodes.
White test item remnants were present on the dorsal surface of the ears of all animals at 25% and 40% between Days 2 and 5, which did not hamper scoring of the skin reactions.
All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Results Pre-screen test:

At 25% and 40%, no signs of systemic toxicity were noted and only very slight irritation was observed and therefore a 40% concentration was selected as highest concentration for the main study.
White test item remnants were present on the dorsal surface of the ears of all animals between Days 2 and 6, which did not hamper scoring of the skin reactions.

Interpretation of results:

other: Not skin sensitizing

Conclusions:

In an LLNA skin sensitisation study, performed according to OECD429 test guideline and GLP principles, the substance was considered not to be a skin sensitiser, as the SI appeared not to be ≥ 3 when tested up to 40%.

Executive summary:

An LLNA skin sensitisation study was performed according to OECD429 test guideline and GLP principles. Based on the results of a pre-screen test, the test concentrations were selected at 10%, 25% and 40%. The very slight irritation of the ears as shown by all animals treated at 25% and 40% between Days 1 and 3 was considered not to have a toxicologically significant effect on the activity of the nodes. White test item remnants were present on the dorsal surface of the ears of all animals at 25% and 40% between Days 2 and 5, which did not hamper scoring of the skin reactions. All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals. No mortality occurred and no clinical signs of systemic toxicity were observed in the animals. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 40% were 641, 501 and 614 DPM, respectively. The mean DPM/animal value for the vehicle control group was 229 DPM. The SI values calculated for the test item concentrations 10, 25 and 40% were 2.8, 2.2 and 2.7, respectively. As the SI appeared not to be ≥ 3 when tested up to 40%, the substance was considered not to be a skin sensitiser.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

DEREK NEXUS version 6.1 did not yield any alerts for skin sensitization for the test item. Additionally, Pergafast 425 does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitizer. 

 

In an in chemico study, performed according to OECD guideline 442C and in accordance with GLP principles, the reactivity of the test substance towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) was determined to assign the test chemical to one of four reactivity classes used to support the discrimination between skin sensitisers and non skin sensitisers. After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and spectrophotometric detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which assigns the test item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers. Dimethylsulfoxide:Acetonitrile (DMSO:ACN, 1:9, v/v); was found to be an appropriate solvent to dissolve the test item and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study. The validation parameters, i.e., calibration curve, mean concentration of Reference Control (RC) samples A, C and C_DMSO:ACN, the CV for RC samples B and C, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the peptide depletion for the test item, were all within the acceptability criteria for the DPRA as stated in the OECD 442C guideline. Upon preparation as well as after incubation of the SPCC and SPCL test item samples, a precipitate was observed.In the cysteine reactivity assay the test item showed 2.8% SPCC depletion while in the lysine reactivity assay the test item showed 0.1% SPCL depletion. The mean of the SPCC and SPCL depletion was 1.5% and as a result the test item was considered to be negative in the DPRA and classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. In conclusion, since all acceptability criteria were met this DPRA is considered to be valid. Pergafast 425 was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since precipitation was observed after the incubation period for both SPCC and SPCL, one cannot be sure how much test item remained in the solution to react with the peptides. Consequently, this negative result is uncertain and should be interpreted with due care. The percentages of SPCC and SPCL depletion might be underestimated.

 

A KeratinoSensTM assay was performed with Pergafast 425 according to OECD guideline 442D and in accordance with GLP principles. The highest test concentration of 125 μM was selected based on solubility. No precipitate was observed at any dose level tested. Two independent experiments were performed. Based on the negative and positive controls it was concluded that the test conditions were adequate and that the test system functioned properly. Pergafast 425 showed no toxicity (no IC₃₀ and IC₅₀ value) and no biologically relevant induction of the luciferase activity (no EC_1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (I_max) was 1.25-fold and 1.09-fold in experiment 1 and 2 respectively. The test item is classified as inconclusive in the KeratinoSens^TM assay since negative results (<1.5-fold induction) were observed at test concentrations < 1000 µM. The test item cannot be classified as negative or positive (based on the absence of a biologically relevant activation of the antioxidant/electrophile responsive element
(ARE)-dependent pathway in keratinocytes at test concentrations < 1000 µM). In conclusion, Pergafast 425 is classified as inconclusive (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

 

The DEREK assessment triggered no alert for skin sensitization for Pergafast 425 and predicted the substance to be not skin sensitizing. PERGAFAST 425 was negative in the DPRA but SPCC or SPCL depletion might be underestimated as precipitation was observed (Key event 1 in the Adverse Outcome Pathway - AOP). In the KeratinoSensTM assay Pergafast 425 is classified as inconclusive based on the absence of a biologically relevant activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes at test
concentrations < 1000 μM (Key event 2 in the AOP). The available results indicate that the in vitro assays are not suitable for the evaluation of Pergafast 425 due to limited solubility.
Performance of a U-SENSTM assay (Key event 3 in the AOP) would, irrespective of the outcome, not yield sufficient information for an overall conclusion regarding skin sensitization.
As the current evidence would not be acceptable for classification and risk assessment and in view of the contention that further in vitro testing would also not lead to a definitive conclusion, it is considered scientifically justified to conduct an in vivo study to assess the potential for skin sensitization of Pergafast 425.

 

An LLNA skin sensitisation study was performed according to OECD429 test guideline and GLP principles. Based on the results of a pre-screen test, the test concentrations were selected at 10%, 25% and 40%. The very slight irritation of the ears as shown by all animals treated at 25% and 40% between Days 1 and 3 was considered not to have a toxicologically significant effect on the activity of the nodes. White test item remnants were present on the dorsal surface of the ears of all animals at 25% and 40% between Days 2 and 5, which did not hamper scoring of the skin reactions. All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals. No mortality occurred and no clinical signs of systemic toxicity were observed in the animals. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 40% were 641, 501 and 614 DPM, respectively. The mean DPM/animal value for the vehicle control group was 229 DPM. The SI values calculated for the test item concentrations 10, 25 and 40% were 2.8, 2.2 and 2.7, respectively. As the SI appeared not to be ≥ 3 when tested up to 40%, the substance was considered not to be a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results the substance does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact in accordance with Regulation (EC) No 1272/2008 and its amendments.