2,6,6-trimethylbicyclo[3.1.1]heptane-3-carbaldehyde

Endpoint summary

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 8 – 9 weeks old
- Housing: group housing (5 animals per group) in Makrolon Type III cages, with wire mesh top
- Diet: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Relative Humidity (%): approx. 45 - 65
- Photoperiod (hrs dark / hrs light): 12 / 12 (6.00 p.m. - 6.00 a.m. / 6.00 a.m. - 6.00 p.m.)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

5% (w/w), 10% (w/w), 25% (w/w)

No. of animals per dose:

5 females

Details on study design:

RANGE FINDING 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 100% of the undiluted test item. Test item solution at different concentrations was prepared using acetone:olive oil (4+1 v/v) as vehicle. Vortexing was used to formulate the test item.
To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed in two animals. Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 100% (undiluted test item) and 50% 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. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (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.
At the tested concentrations the animals did not show any signs of systemic toxicity. On days 3 to 6, both animals showed an erythema of the ear skin (for the animal treated with 50% of the test item: score 2 for days 3 to 5 and score 1 for day 6; for the animal treated with the undiluted test item: score 2 for days 3 to 6). Increase in ear weights was 32.5% and 23.2%, respectively, compared to historical vehicle values. As the ear weight for the animal treated with 50% test item exceeded the threshold value of 25%, a second pre-test was performed using test item concentrations of 10 and 25%, respectively.
In the second pre-test, the animal treated with 25% of the test item showed an erythema of the ear skin (score: 1 on days 3 to 6). No local effects were observed with 10% of the test item. Ear weights and increase in ear thickness over time were both within the range of <25% increase.
Thus, the test item in the main study was assayed at 5, 10, and 25%. The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiments.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- 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:
The test item was placed into an appropriate container on a tared balance and acetone:olive oil (4+1 v/v) was added. 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.
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with test item concentrations of 5, 10, and 25% (w/w) in AOO (acetone/olive oil (4:1 v/v)). The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (Ø ~ 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 (control animals). Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline containing 20.2 µCi of ³H-methyl thymidine (equivalent to 80.6 µCi/mL ³HTdR) were injected into each test and control mouse via the tail vein.

DETERMINATION OF INCORPORATED ³HTdR:
Approximately five hours after treatment with 3HTdR all mice were euthanised 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 at least 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 ß-scintillation counter. Similarly, background ³HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The ß-scintillation counter expresses ³HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

DETERMINATION OF LYMPH NODE WEIGHT AND CELL COUNT:
After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance. Furthermore, the lymph node cell count was determined for each animal. For this, the volume of the cell suspensions was adjusted to an equal final volume and vortexed. Subsequently, individual cell counts were determined using a cell counter (CAS 1, Schärfe System). The values obtained were taken down manually.

DETERMINATION OF EAR WEIGHTS:
After the lymph nodes had been excised, both ears (left and right) of mice were punched at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm²). For each animal both punches were immediately weighed (pooled per animal) using an analytical balance.

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, 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 the test item groups and negative control group. For all statistical calculations SigmaStat for Windows (Version 2.0) was used. A One-Way-Analysis-of-Variance was used as a 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 percent level (p < 0.05).
The Dean-Dixon-Test was used for detection of possible outliers (performed with Microsoft Excel 2007). However, both biological and statistical significance were considered together.
Where appropriate, the EC3 value was calculated according to the equation EC3 = (a-c) [(3-d)/(b-d)] + c, where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.

Parameter:

SI

Remarks on result:

other: Stimulation Indices (S.I.) of 1.28, 2.04, and 3.80 were determined with the test item at concentrations of 5, 10, and 25 % (w/w) in acetone:olive oil (4+1 v/v), respectively, and an EC3 value of 18.2 % (w/w) was calculated.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: The DPM values showed a statistically significant and biologically relevant increase in all dose groups in comparison to the vehicle control group and furthermore, a clear dose dependent increase in DPM value was observed.

Calculation of Stimulation Indices per Dose Group:

Test item concentration	Group Calculation	SD	S.I.
	Mean DPM per animal (2 lymph nodes)^a)
Vehicle Control (acetone:olive oil (4+1 v/v))	266.1	131.0	1.00
5% test item	340.7	144.1	1.28
10% test item	543.5*	248.4	2.04
25% test item	1010.5*	482.3	3.80

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)

*   Mean DPM value for the group was according to the ANOVA (Dunnett-test) significantly higher than the corresponding conttrol value (p<0.05).

Calculation of the EC3 value:

	Test item concentration %	S.I.
Test Group 2	10 (a)	2.04 (b)
Test Group 3	25 (c)	3.80 (d)
EC3 = (a-c) [(3-d)/(b-d)] + c = 18.2% (w/w)

EC3 = Estimated concentration for a S.I. of 3.

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.

- Viability/Mortality: No deaths occurred during the study period.

- Clinical Signs: No signs of systemic toxicity or local skin erythema were observed during the study period.

- Body Weights: The body weight of the animals, recordedprior to the first application and prior to treatment with³HTdR, was within the range commonly recorded for animals of this strain and age.

- Lymph Node Weights and Cell Counts: The measured lymph node weights and –cell counts of all animals treated was recorded after sacrifice. A statistically significant or biologically relevant increase in lymph node weights was not observed in any of the test item treated groups in comparison to the vehicle control group. A statistically significant and biologically relevant increase in lymph node cell counts (p <0.05) was observed for the high dose group in comparison to the vehicle control group. For BALB/c mice, a cutoff value for the lymph node cell count index of 1.55 was reported for a positive response. The index determined for the lymph node cell count of the high dose group exceeded this threshold.

- Ear Weights: The measured ear weight of all animals treated was recorded on test day 6 (after necropsy). A biologically relevant or statistically significant increase in ear weights was not observed. Furthermore, the cutoff-value (1.1) of the ear weight index for a positive response regarding ear skin irritation reported for BALB/c mice was not exceeded in any of the treated groups.

Interpretation of results:

sensitising

Remarks:

Migrated information

Conclusions:

The test item 3-Formylpinan was found to be a skin sensitiser under the test conditions of this study.

Executive summary:

The study was performed according to OECD guideline 429 in compliance with GLP.

In this study the test item 3-Formylpinan was assessed for its skin sensitising potential using the Local Lymph Node Assay (LLNA) in mice. Test item solution at different concentrations was prepared in the vehicle acetone:olive oil (4+1 v/v).

The local lymph node assay is recommended by international test guidelines (e.g., OECD) as an animal test for predicting skin sensitisation in humans and provides a rational basis for risk assessment. The basic principle underlying the LLNA is that sensitisers induce a primary proliferation of lymphocytes in the lymph node draining the application site. The ratio of proliferation in test item treated groups compared to that in vehicle controls is termed the Stimulation Index (S.I.). Radioactive labeling is used to measure cell proliferations.

For this purpose a local lymph node assay was performed using test item concentrations of 5, 10, and 25% (w/w). The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation (as determined by two pre-experiments).

The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed.

No statistically significant increase in ear weights was observed in the treatment groups in comparison to the vehicle control group. Furthermore, for BALB/c mice, a cutoff value of 1.1 for the ear weight index was reported for a positive response regarding ear skin irritation. None of the indices determined for the test item treated groups exceeded this threshold.

A test item is regarded as a sensitiser in the LLNA if exposure to one or more test item concentration results in a 3-fold or greater increase in incorporation of ³HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated test item concentration required to produce a S.I. of 3 is referred to as the EC3 value.

In this study Stimulation Indices (S.I.) of 1.28, 2.04 and 3.80 were determined with the test item at concentrations of 5, 10, and 25% (w/w) in acetone:olive oil (4+1 v/v), respectively. A clear dose response was observed.

A statistically significant increase in DPM value (p≤ 0.05) was observed in the animals treated with test item concentrations of 10% and 25%, respectively. The increase obtained with 25% (high dose) was considered to be biologically relevant. Furthermore, a statistically significant and biologically relevant increase in lymph node cell count was observed in the highest dose group in comparison to the vehicle control group (p≤ 0.05). For BALB/c mice, a cutoff value for the lymph node cell count index of 1.55 was reported for a positive response. According to this criterion, the lymph node cell count index determined for the high dose group (2.51) indicates a positive response. A statistically significant increase in lymph node weights was not observed in any of the treatment groups in comparison to the vehicle control group.

The test item 3-Formylpinan was found to be a skin sensitizer and an EC3 value of 18.2 % was derived.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

In vivo:

The key study was performed according to OECD guideline 429 in compliance with GLP (BASF SE, 2013). In order to study a possible skin sensitising potential of 3-Formylpinan, three groups each of five female mice were treated once daily with the test item at concentrations of 5, 10, and 25% in acetone:olive oil (4+1 v/v) by topical application to the dorsum of each ear for three consecutive days. The test item could be dissolved in the vehicle. The appropriateness of the used concentrations was previously assessed in two pre-experiments.

A control group of five mice was treated with the vehicle acetone:olive oil (4+1 v/v) 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, pooled per animal and immediately weighed. Furthermore, after excision of the lymph nodes, both ears of the mice were punched at the apical area using a biopsy punch and were immediately weighed pooled per animal using an analytical balance. Afterwards single cell suspensions of lymph node cells were prepared from pooled lymph nodes per animal. An aliquot of each cell suspension was used for determination of lymph node cell count. Subsequently the suspensions were 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 ab-scintillation counter.

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. No statistically significant increase in ear weights was observed in the treatment groups in comparison to the vehicle control group. Furthermore, for BALB/c mice, a cutoff value of 1.1 for the ear weight index was reported for a positive response regarding ear skin irritation (see Ref. 9). None of the indices determined for the test item treated groups exceeded this threshold.

A test item is regarded as a sensitiser in the LLNA if exposure to one or more test item concentration results in a 3-fold or greater increase in incorporation of³HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated test item concentration required to produce a S.I. of 3 is referred to as the EC3 value.

In this study Stimulation Indices (S.I.) of 1.28, 2.04 and 3.80 were determined with the test item at concentrations of 5, 10, and 25% in acetone:olive oil (4+1 v/v), respectively. A dose response was observed.

A statistically significant increase in DPM value (p≤ 0.05) was observed in the animals treated with test item concentrations of 10% and 25%, respectively. The increase obtained with 25% (high dose) was considered to be biologically relevant. Furthermore, a statistically significant and biologically relevant increase in lymph node cell count was observed in the highest dose group in comparison to the vehicle control group (p≤ 0.05). For BALB/c mice, a cutoff value for the lymph node cell count index of 1.55 was reported for a positive response. According to this criterion, the lymph node cell count index determined for the high dose group (2.51) indicates a positive response. A statistically significant increase in lymph node weights was not observed in any of the treatment groups in comparison to the vehicle control group. An EC3 value of 18.2% (w/w) could be derived.

Conclusion: The test item 3-Formylpinan was found to be a skin sensitiser under the test conditions of this study.

In vitro:

A combination of several in-house validated in vitro methods addressing major steps of the skin sensitization process (protein reactivity, activation of keratinocytes and activation of dendritic cells) has been conducted to assess the skin sensitizing potential of 3-Formylpinan. The individual assays were combined into a strategy to qualitatively assess the skin sensitizing potential of 3-Formylpinan in vitro.

A Direct Peptide Reactivity Assay (DPRA) has been performed in compliance with GLP (BASF SE, 64V0593/00A028, 2013). The reactivity of the test substance towards proteins with nucleophilic side chains such as cysteine or lysine residues was evaluated.

For this purpose the test item was incubated with synthetic peptides in ratios of 1:10 (for C-peptide) or 1:50 (for K-peptide) for approx. 24 hours at room temperature. Additionally, vehicle controls and co-elution controls were analyzed. The remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 258 nm.

The mean C-peptide depletion caused by 3-Formylpinan was determined to be 39.8%. The mean K-peptide depletion caused by the test substance was determined to be 4.6%. Thus, the mean peptide depletion was calculated to be 22.2%.

Based on the observed results and applying the prediction model proposed in Gerberick et al. (2007) it was concluded that 3 -Formylpinan shows a low chemical reactivity in the DPRA under the test conditions chosen. According to the classification tree model described by Gerberick et al. 3 -Formylpinan is predicted to be a sensitizer.

 

The myeloid U937 skin sensitization test (MUSST) is a dendritic cell activation test to predict skin sensitizing potential and was performed in compliance with GLP (BASF SE, 65V0593/00A029, 2013). The human pro-monocytic cell line U937 was used as model for dendritic cells. After 48 hours of test substance exposure using concentrations between 2.03 and 32.40 μg/mL, the cell membrane marker CD86 was quantitatively detected using flow cytometry. After 48 hours of exposure to the test substance CD 86 expression was not induced in U937 cells affording at least 70% viability in two independent experiments. From this it has to be concluded that 3 -Formylpinan does not induce dendritic cell activation.

The ARE Reporter Assay (LuSens), i.e. a keratinocyte activation assay, was performed with 3-Formylpinan in compliance with GLP (BASF SE, 66V0593/00A030, 2013). LuSens cells, a transgenic keratinocyte cell line derived from HaCaT cells, were incubated with 3 -Formylpinan using six concentrations (6.88 up to 35.48 μg/mL, in culture medium containing 1% DMSO). After 48 hours, luciferase activity was measured as read out for the respective reporter gene activation via the Keap1/Nrf2/ARE signaling pathway. In parallel a MTT assay was performed to assess cytotoxicity. In total eight independent valid experiments were conducted and luciferase activities just above 1.5-fold at test substance concentrations that did not reduce cell viability below 70% were also observed. However, the results could not be clearly confirmed in following experiments at non-cytotoxic concentrations. Hence, the keratinocyte activating potential of 3-Formylpinan could not be conclusively evaluated.

 

The individual test methods, the evaluation criteria and the experimental results are summarized in the following table:

Test method	Endpoint	Evaluation criteria	Experimental result	Test result
Direct Peptide Reactivity Assay (DPRA)	Peptide depletion	positive if ≥6.38% mean peptide depletion; negative if <6.38% mean peptide depletion	22.2% mean peptide depletion	positive
Keratinocyte Activation Assay-LuSens	Luciferase activity	positive if ≥1.5-fold luciferase activity when viability is >70%of the vehicle control; negative if <1.5-fold luciferase activity	Ambiguous results concerning activation of keratinocytes in eight independent experiments	inconclusive
Dendritic Cell Line Activation Assay Myeloid U937 Skin Sensitization Test (MUSST)	CD86 expression	positive if ≥1.2-fold of CD86 when viability is >70% of the control; negative if <1.2-fold of CD86	No induction of CD 86 expression	negative

 

Taking the individual and combined sensitivities and specificities of the three in vitro assays into account the decision matrix below is used to predict the skin sensitizer potential of the test substance:

DPRA	LuSens	MUSST	Test strategy prediction
positive	positive	positive	sensitizer
positive	positive	negative	sensitizer
positive	negative	positive	sensitizer
positive	negative	negative	non-sensitizer
negative	positive	positive	sensitizer
negative	positive	negative	non-sensitizer
negative	negative	positive	non-sensitizer
negative	negative	negative	non-sensitizer

 

Based on the results summarized in the table above and applying the evaluation criteria, 3-Formylpinan is peptide reactive but does not activate dendritic cells. However, the keratinocyte activating potential of 3-Formylpinan could not be conclusively evaluated. Taken together, the sensitization potential of 3-Formylpinan could not be predicted using the in vitro test battery on skin sensitization.

Migrated from Short description of key information:
Mouse local lymph node assay (LLNA): sensitising
In vitro test battery on skin sensitization (DPRA, MUSST, LuSens): no prediction possible

Justification for selection of skin sensitisation endpoint:
GLP guideline study. No prediction concerning skin sensitisation possible based on results from in vitro studies.

Respiratory sensitisation

Endpoint conclusion

Additional information:

No data available.

Justification for classification or non-classification

The present data on dermal sensitization fulfill the criteria laid down in 67/548/EEC and regulation (EU) 1272/2008. Therefore, a classification with R43 and "Skin sensitisation" (Category 1B) is warranted.