Reaction mass of hentriacontan-16-one and pentatriacontan-18-one and tritriacontan-16-one

Endpoint summary

Description of key information

Skin sensitisation:

One in vitro and one in chemico skin sensitisation study (DPRA assay and KeratinoSensTM assay) are available, since no suitable solvent could be found to dissolve the test item at the desired concentration, no main experiment could be performed. The third in vitro assay, the U-SENSTM assay, was not done as the same solvents are to be used as for the KeratinosensTM assay.

One in vivo study (LLNA) is available which was based on OECD No.429 (2010) and resulted the substance is not a skin sensitizer.

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:

From 2017-09-21 to 2017-10-24

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:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

Batch No: FK650468
Purity: not speific

Species:

mouse

Strain:

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
- Condition: Inbred, SPF-Quality
- Age at study initiation: Young adult animals (approximately 11 weeks old)
- Weight at study initiation: 20.9 to 23.9 g.
- Housing: 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 sawdust as bedding material equipped with water bottles
- Diet (e.g. ad libitum): Pelleted rodent diet, ad libitum
- Water (e.g. ad libitum): Municipal tap-water, freely available
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C
- Humidity (%): 45 to 71%
- Air changes (per hr): Ten or greater air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle
- IN-LIFE DATES: From: 2017-09-27 To: 2017-10-24

Vehicle:

methyl ethyl ketone

Concentration:

5%, 10%, 20%

No. of animals per dose:

Five females per group.

Details on study design:

PRE-SCREEN TESTS:
- Irritation: Not observed
- Systemic toxicity: Not observed
- Ear thickness measurements: Variations in ear thickness during the observation period were less than 25% from Day 1 predose values.
- Erythema scores: 0

MAIN STUDY
Three groups of five animals were treated with one test item concentration per group. The highest test item concentration was selected from the pre-screen test. One group of five animals was treated with the vehicle.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer

TREATMENT PREPARATION AND ADMINISTRATION:
- Induction - Days 1, 2 and 3: The dorsal surface of both ears was topically treated (25 μL/ear or an equivalent amount when dosed with a spatula) with the test item, at approximately the same time on each day. The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item.
- Excision of the Nodes - Day 6: Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) containing 20 μCi of 3H-methyl thymidine.
After five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) of Euthasol® 20%. The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in PBS.
- Tissue Processing for Radioactivity - Day 6: Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (maze size: 200 μm, diameter: ± 1.5 cm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) and then stored in the refrigerator until the next day.
- Radioactivity Measurements - Day 7: Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter. Counting time was to a statistical precision of ±0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

Parameter:

SI

Value:

1.2

Test group / Remarks:

at 5% concentrations

Parameter:

SI

Value:

0.9

Test group / Remarks:

at 10% concentrations

Key result

Parameter:

SI

Value:

1.3

Test group / Remarks:

at 20% concentrations

Cellular proliferation data / Observations:

EC3 CALCULATION
Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 20%, the test item was not considered to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 20%.
CLINICAL OBSERVATIONS:
- Skin Reactions / Irritation: No irritation was observed in any of the animals. White test item remnants were present on the dorsal surface of the ears of all test item treated animals between Days 1 and 5, which did not hamper scoring of the skin reactions.
- Systemic Toxicity: No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.
- Macroscopic Examination of the Lymph Nodes and Surrounding Area: 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.
- Radioactivity Measurements: Mean DPM/animal values for the experimental groups treated with test item concentrations 5, 10 and 20% were 150, 114 and 158 DPM, respectively. The mean DPM/animal value for the vehicle control group was 126 DPM.

Interpretation of results:

GHS criteria not met

Conclusions:

Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 20%, the test item was not considered to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 20%.
Based on these results, the test item would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines.

Executive summary:

The objective of this study was to evaluate whether test item induces skin sensitization in mice after three epidermal exposures of the animals based on OECD No.429 (2010).

Test item concentrations selected for the main study were based on the results of a pre-screen test.

In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 5, 10 or 20% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (methylethylketone). Three days after the last exposure, all animals were injected with 3Hmethyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

No irritation was observed in any of the animals.

The mean body weight gain shown by the animals over the study period was considered to be normal.

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.

Mean DPM/animal values for the experimental groups treated with test item concentrations 5, 10 and 20% were 150, 114 and 158 DPM, respectively. The mean DPM/animal value for the vehicle control group was 126 DPM. The SI values calculated for the test item concentrations 5, 10 and 20% were 1.2, 0.9 and 1.3, respectively.

Since there was no indication that the test item elicits a SI≥3 when tested up to 20%, test item was not considered to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 20%.

Based on these results, test item would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 2017-08-10 to 2017-08-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

Batch No: FK650468
Purity: not speific

Details on the study design:

- Preparation of Test Item Stock, Spiking and Working Solutions
No correction was made for the composition/purity of the test item.
A solubility test was performed. The test item was suspended in Dimethyl sulfoxide (DMSO) to a final concentration of 40 mg/mL. The suspension was vortexed and sonicated (56 minutes with a maximum temperature of 37°C). The test item did not dissolve and floated on the DMSO solution. It was also tried to dissolve the test item at concentrations of 20, 10, 2.5 mg/mL. The suspensions were vortexed and sonicated. The test item did not dissolve in DMSO at any of these concentrations (clear non-homogeneous suspensions with test item lumps). The solubility at 2.5 mg/mL did not improve compared to the solubility at 40 mg/mL.
Next it was tried to dissolve the test item in milli-Q water at 40, 20 and 2.5 mg/mL. The test item formed suspensions which were vortexed and sonicated. The test item did not dissolve in milli-Q water at any of these concentrations (clear non-homogeneous suspensions with test item lumps). The solubility at 2.5 mg/mL did not improve compared to the solubility at 40 mg/mL.
Any residual volumes were discarded.

Remarks on result:

not determinable

The test item did not dissolve in DMSO and Milli-Q water. The test item floated on the surface or formed a suspension with lumps (non-homogeneous). Lowering test item concentrations from 40 mg/mL to 2.5 mg/mL did not improve the solubility.

Interpretation of results:

study cannot be used for classification

Conclusions:

A solubility test was performed. No suitable solvent could be found to dissolve the test item at the desired concentration. Test itrm was therefore considered to be not suitable for testing in the KeratinoSensTM assay. No main assay was performed.

Executive summary:

To evaluate the ability of test item to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSensTM assay according to OECD guideline 442D.

A solubility test was performed. No suitable solvent could be found to dissolve the test item at the desired concentration. The test item was therefore considered to be not suitable for testing in the KeratinoSensTM assay. No main assay was performed.

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-08-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes

Type of study:

other: Direct Peptide Reactivity Assay (DPRA)

Specific details on test material used for the study:

Batch No: FK650468
Purity: not speific

Details on the study design:

Solubility of the test item in an appropriate solvent was assessed before performing the DPRA. An appropriate solvent dissolved the test item completely, i.e. by visual inspection the solution had to be not cloudy nor have noticeable precipitate. The following solvents were evaluated: acetonitrile (ACN), Milli-Q water (MQ), MQ/ACN (1:1, v/v), isopropanol, acetone, acetone/ACN (1:1, v/v) and dimethylsulfoxide (DMSO)/ACN (1:9, v/v).

Remarks on result:

not determinable

Other effects / acceptance of results:

At a concentration of 100 mM, the test item was not soluble in ACN, MQ, MQ/ACN (1:1, v/v), isopropanol, acetone, acetone/ACN (1:1, v/v) and DMSO/ACN (1:9, v/v). Therefore no DPRA study could be performed and the study was stopped.

Interpretation of results:

study cannot be used for classification

Conclusions:

In conclusion, since no suitable could be found that could be used to dissolve the test item at the desired concentration and was compatible with the DPRA, no main experiment could be performed. As a result, the reactivity of test item towards SPCC and SPCL could not be determined.

Executive summary:

The reactivity of test item towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) was determined according to OECD guideline 442C.

However, since no suitable solvent could be found to dissolve the test item at the desired concentration that was also compatible with the Direct Peptide Reactivity Assay (DPRA), the reactivity of test item towards SPCC or SPCL could not be determined.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Performance of a DPRA assay and KeratinoSensTM assay was attempted in accordance with Section 8.3 of Annex VII of Regulation (EC) No 1907/2006 as amended in Commission Regulation (EU) 2016/1688 of 20 September 2016 and the strategy presented in ECHA Guidance on information requirements and chemical safety assessment Chapter R.7a.

No suitable solvent could be found to dissolve the test item at a desired concentration. The test item was therefore considered to be not suitable for testing in the DPRA assay or in the KeratinoSensTM assay. No main assays were performed.

Performance of a third in vitro assay, the U-SENSTM assay, was not done as the same solvents are to be used as for the KeratinosensTM assay. Furthermore, the endpoint conclusion cannot be drawn based on the outcome of a single in vitro assay. Therefore it was considered scientifically justified to omit further in vitro testing and to proceed with in vivo testing to determine skin sensitizing properties of the test item.

The in vivo LLNA study was conductedd to evaluate whether test item induces skin sensitization in mice after three epidermal exposures of the animals based on OECD No.429 (2010).

Three experimental groups of five female CBA/J mice were treated with test item concentrations of 5, 10 or 20% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (methylethylketone). Three days after the last exposure, all animals were injected with 3Hmethyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

No irritation was observed in any of the animals.

The mean body weight gain shown by the animals over the study period was considered to be normal.

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.

Mean DPM/animal values for the experimental groups treated with test item concentrations 5, 10 and 20% were 150, 114 and 158 DPM, respectively. The mean DPM/animal value for the vehicle control group was 126 DPM. The SI values calculated for the test item concentrations 5, 10 and 20% were 1.2, 0.9 and 1.3, respectively.

Since there was no indication that the test item elicits a SI≥3 when tested up to 20%, test item was not considered to be a skin sensitizer.

Justification for classification or non-classification

Skin sensitisation:

Negative result in animal test (LLNA).

According to Regulation (EC) No 1272/2008, table 3.4.2, this substance is not classified for this endpoint.