Benzenepropanoic acid, 3,5-bis(1,1-dimethylethyl)-4-hydroxy-, 2-ethylhexyl ester

Endpoint summary

Description of key information

Contradicting data are available. The test article was sensitizing in an LLNA, while the strucural analogue substance did not cause skin sensitization in the guinea pig maximization test (OECD 406, GLP). Furhter studies are ongoing to better understand this issue.

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:

weight of evidence

Study period:

04 Dec 2017 - 02 Aug 2018

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)

Qualifier:

according to guideline

Guideline:

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

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: Envigo RMS GmbH, C/O Postfach 553, NL-5800 AN Venray
- Age at study initiation: 9 weeks (pretest), 8 weeks (main test)
- Weight at study initiation: 19.1 g – 20.9 g (pretest), 17.9 g – 21.4 g (main test)
- Housing: single housing, Polycarbonate cages type MII with mesh wire tops supplied by BECKER & Co., Castrop-Rauxel, Germany
- Diet: Kliba mouse/rat maintenance diet “GLP” supplied by Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum.
- Water: Drinking water ad libitum
- Acclimation period: at least 5 days
- Indication of any skin lesions:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 24°C
- Humidity (%): 45-65 %
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

methyl ethyl ketone

Concentration:

10%, 25% and 50%

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
A solubility experiment was performed according to the recommendations given by OECD 429. The highest test-substance concentration that was used in the pretest was a 50% testsubstance preparation. In order to determine the highest test-substance concentration that does not induce local signs of skin irritation and/or systemic toxicity, a pretest (experimental conduct in accordance with GLP, but without a GLP status) was performed. Two mice were treated with test-substance concentrations of 10% and 50% each on three consecutive days. Clinical signs were recorded after each application as well as on day 5. Signs of local irritation were recorded on day 1, 2 and 5. Prior to the first application of the test item (day 0), on day 2 and before sacrifice (day 5) the ear thickness was determined using a micrometer. Furthermore, the ears were punched after sacrifice at the apical area using a biopsy punch (Ø 0.8 cm) and were immediately pooled per animal and weighed using an analytical balance. Additionally, the weight of the pooled lymph nodes from both sides was determined for each animal. No signs of systemic toxicity were observed in the pretest. At the tested concentrations, the animals did not show relevant signs of local irritation as confirmed by determination of the ear weight (compared to current historical vehicle values) and ear thickness measurements. However, the 50% concentration showed considerably increased lymph node weights. Therefore, the following dose levels were selected for the main study: 10%, 25% and 50% (w/w).

MAIN STUDY
Prior to first application, the animals were distributed to the individual groups, received animal numbers and were allocated to the respective cages according to the randomization instructions of „Nijenhuis, A. and Wilf, H.S.: Combinatorial Algorithms, Academic Press, New York, San Francisco, London, 1978, pp. 62 – 64“. Individual body weights on day 0 prior to the first application and on day 5 prior to the sacrifice of the animals. Obvious signs of systemic toxicity and/or local inflammation at the application sites were noted for each animal in the raw data. A check for moribund and dead animals was made twice each workday (beginning and end) and once on Saturdays, Sundays and on public holidays. Epicutaneous application is simulating dermal contact with the compound, which can occur under practical use conditions. Application volume: 25 μL per ear. Site of application: Dorsal surface of both ears Frequency of application: 3 consecutive applications (day 0 – day 2) to the same application site. On study day five (about 66 to 72 hours after the last application of test substance to the ears), 20 μCi 3H-thymidine in 250 μL sterile saline were injected into the tail vein of the mice. The animals were sacrificed on study day 5 about 5 to 6 hours after 3H-thymidine injection by
cervical dislocation under Isoflurane anesthesia. Immediately after the death of each animal, a circular piece of tissue (diameter 0.8 cm) was punched out of the apical part of each ear of all animals. The weight of the pooled punches was determined for each animal. These measurements serve for detecting a potential inflammatory ear swelling. Immediately after removal of the ear punches, the left and right auricular lymph nodes were dissected. The weight of the pooled lymph nodes from both sides was determined for each animal. After weight determination, the pooled lymph nodes of each animal were stored in phosphate-buffered saline (PBS) in an ice water bath until further preparation. A single cell suspension was prepared as soon as possible after dissection by carefully passing the lymph nodes through an iron mesh (mesh size 200 μm) into 6 mL phosphate-buffered physiological saline. For determination of cell counts, an aliquot of each suspension was further diluted with Casy®ton in a ratio 1:500. The cell count was determined using a Casy® Counter. The remaining cell suspensions were washed twice with PBS and precipitated with 5% trichloro-acetic acid (TCA). Each precipitate was transferred to scintillation fluid and incorporation of 3H-thymidine into the cells was measured in a β-scintillation counter.

Positive control substance(s):

other: Studies using the positive control substance Alpha-Hexylcinnamaldehyde, techn. 85%, are performed twice a year in the laboratory to show that the test system is able to detect sensitizing compounds under the test conditions chosen (reliability check).

Statistics:

Mean values and standard deviations of the measured parameters were calculated for the test and control groups from the individual values. The stimulation indices of 3H-thymidine incorporation, cell count, lymph node weight and ear weight measurements were calculated as the ratio of the test group mean values for these parameters divided by that of the vehicle control group. The ³H-thymidine incorporation, cell count, lymph node weight and ear weight was statistically analyzed with the WILCOXON - Test.

Parameter:

EC3

Remarks:

(%)

Value:

18.8

Parameter:

SI

Value:

1.94

Test group / Remarks:

10 %

Parameter:

SI

Value:

3.74

Test group / Remarks:

25 %

Parameter:

SI

Value:

7.08

Test group / Remarks:

50 %

Cellular proliferation data / Observations:

When applied as 25% and 50% preparation in MEK, the test substance induced a biologically relevant (increase to 3-fold or above of control value = stimulation index (SI) ≥ 3), statistically significant and concentration-dependent increase of 3H-thymidine incorporation into the auricular lymph node cells. Concomitantly, the 25% and 50% test-substance preparation induced a biologically relevant, statistically significant and concentration-dependent response (increase to 1.5-fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts. In addition, statistically significant, biologically relevant and concentration-dependent increase in lymph node weights was observed at the 25% and 50% concentration. The increase at the 10% concentration was also statisticially significant.

The test-substance concentrations did not cause increases (SI ≥ 1.25) in ear weights demonstrating the absence of relevant ear skin irritation. The increases at the 25 and 50% test substance preprations were, however, statistically significant. The expected body weight gain was generally observed during the study. No signs of systemic toxicity were noticed in all animals during general observation. No local findings were observed during the observation period.

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

It is concluded that the test article exhibits a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen.

Executive summary:

The skin sensitizing potential of the test article was assessed using the radioactive Murine Local Lymph Node Assay. Groups of 5 female CBA/CaOlaHsd mice each were treated with 10%, 25% and 50% (w/w) preparations of the test substance in MEK (methyl ethyl ketone) or with the vehicle alone. The study used 3 test groups and 1 control group. Each test animal was treated with 25 μL per ear of the appropriate test-substance preparation applied to the dorsal surfaces of both ears on three consecutive days. The control group was treated with 25 μL per ear of the vehicle alone. Three days after the last application, 20 μCi 3H-thymidine in 250 μL sterile saline were injected into the tail vein of the mice. About 5 to 6 hours after the 3H-thymidine injection, the mice were sacrificed and the auricular lymph nodes were removed. Lymph node response was evaluated measuring 3H-thymidine incorporation (indicator of cell proliferation). Cell counts and weights of each animal’s pooled lymph nodes were also determined. In addition, a 0.8 cm diameter sample was punched out of the apical part of each ear, and for each animal, the weight of the pooled punches was determined to obtain an indication of possible skin irritation. No signs of systemic toxicity were noticed in all animals during general observation. When applied as 25% and 50% preparation in MEK, the test substance induced a biologically relevant (increase to 3-fold or above of control value = stimulation index (SI) ≥ 3), statistically significant and concentration-dependent increase of 3H-thymidine incorporation into the auricular lymph node cells. Concomitantly, the 25% and 50% test-substance preparation induced a biologically relevant, statistically significant and concentration-dependent response (increase to 1.5-fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts. In addition, statistically significant, biologically relevant and concentration-dependent increase in lymph node weights was observed at the 25% and 50% concentration. The increase at the 10% concentration was also statisticially significant. The test-substance concentrations did not cause increases (SI ≥ 1.25) in ear weights demonstrating the absence of relevant ear skin irritation. The increases at the 25% and 50% test-substance preprations were statistically significant. Thus, it is concluded that the test item exhibits a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen. The threshold concentration for sensitization induction was >10% <25%. The EC 3 (estimated concentration that leads to the SI of 3.0) for 3H-thymidine incorporation and the EC 1.5 (estimated concentration that leads to the SI of 1.5) for cell count was calculated by linear regression from the results of these concentrations to be 18.8% and 18.4%, respectively.

Reference 2

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

July 03rd 1989, August 03rd 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

1981

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The study was conducted in 1989 when the GPMT was an internationally accepted and recommended method.

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Animal strain as given in the report: Pirbright White Strain (Tif: DHP)
- Source: CIBA-GEIGY Limited, Tierfarm, 4332 Stein / Switzerland
- Age at study initiation: approx. 10 weeks old
- Weight at study initiation: 336 to 446 g
- Housing: individually in Macrolon cages (Type 3)
- Diet: standard guinea pig pellets - NAFAG No. 845, Gossau SG, ad libitum
- Water: fresh water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 22+3°C
- Humidity: 30 to 70 %
- Photoperiod (hrs dark / hrs light): 12 hours light cycle day

All batches of the diet are assayed for nutritive ingredients and contamination level by the manufacturer. Analytical results are available at the animal supply office. The drinking water is examined periodically by the IWB (Industrielle Werke Basel).

Route:

intradermal

Vehicle:

other: sesame oil

Concentration / amount:

10 %

Day(s)/duration:

day 0

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Concentration / amount:

10 %

Day(s)/duration:

day 7 / 48 h

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Concentration / amount:

10 %

Day(s)/duration:

day 21 / 24 h

No. of animals per dose:

10 male and 10 female guinea pigs per group

Details on study design:

The following concentrations have been examined on separate animals for the evaluation of the primary irritant potential and the maximum subirritant concentration: 30 and 10 % in vaseline.
Erythema reactions were observed with 30% in vaseline. 30% was, therefore, selected for the epidermal induction application and 10% as maximum subirritant concentration for the epidermal challenge application.

The induction was a two-stage operation. First, intradermal injections (into the neck region); second, closed patch exposure over the injection sites one week later.
First Induction, intradermal application
Three pairs of intradermal injections (0.1 mL per injection) were made simultaneously into the shaved neck of the guinea pigs as follows:
- adjuvant/saline mixture 1:1 (v/v)
- test article in sesame oil (w/v)
- test article in the adjuvant saline mixture (w/v)
Second Induction, epidermal application
One week later the test article was incorporated in vaseline (w/w) and applied on a filterpaper patch to the neck of the animals (patch 2x4 cm; approx. 0.4 g paste per patch; occluded administration for 48 hours).

Challenge
Two weeks after the epidermal induction application the animals were tested on the flank with test material in vaseline (w/w) and the vehicle alone (patch 2x2 cm; approx. 0.2 g paste per patch; occluded administration for 24 hours).

Twenty four hours after removing the dressings, the challenge reactions were graded according to the Draize scoring scale. A second evaluation was made 48 hours after removing the dressings. The sensitising potential of the test item was classified according to the grading of Magnusson and Kligman. The body weight was recorded at start and end of the test.

Observations and records
Induction reactions
After the intradermal and the epidermal induction application irritant reactions are normally induced by the adjuvant, the high test article concentration, or the sodium lauryl sulfate pretreatment. Because most of the reactions are treatment related and not compound related, the reactions are only described in special cases in the section of results.
Challenge reactions
Twenty four hours after removing the dressings, the challenge reactions were graded according to the Draize scoring scale. A second evaluation was made 48 hours after removing the dressings.

The sensitising potential of the test item was classified according to the grading of Magnusson and Kligman. The body weight was recorded at start and end of the test.

Challenge controls:

A control group was treated with adjuvant and the vehicle during the induction period. During the challenge period the group was treated with the vehicle as well as with the test article (at least 10 animals) to check the maximum subirritant concentration of the test article in adjuvant treated animals.

Positive control substance(s):

no

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

10%

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

none

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

10%

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

none

Interpretation of results:

GHS criteria not met

Conclusions:

In the GPMT, none of the guinea pigs of the test group showed skin reactions 24 and 48 hours after challenge with 10% test item.

Reference 3

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

August 25 to September 25, 1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

1981

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The study was conducted in 1987 when the GPMT was an internationally accepted and recommended method.

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Animal strain as given in the report: Pirbright White Strain (Tif: DHP)
- Source: CIBA-GEIGY Limited, Tierfarm, 4332 Stein / Switzerland
- Age at study initiation: approx. 10 weeks old
- Weight at study initiation: 336 to 462 g
- Housing: individually in Macrolon cages (Type 3)
- Diet: standard guinea pig pellets - NAFAG No. 846, Gossau SG, ad libitum
- Water: fresh water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 22+3°C
- Humidity: 30 to 70 %
- Photoperiod: 12 hours light cycle day

All batches of the diet are assayed for nutritive ingredients and contamination level by the manufacturer. Analytical results are available at the animal supply office. The drinking water is examined periodically by the IWB (Industrielle Werke Basel).

Route:

intradermal

Vehicle:

other: sesame oil

Concentration / amount:

3 %

Day(s)/duration:

day 0

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Concentration / amount:

3 %

Day(s)/duration:

day 7

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Concentration / amount:

3 %

Day(s)/duration:

day 21 / 24 h

No. of animals per dose:

10 male and 10 female guinea pigs per group

Details on study design:

The following concentrations have been examined on separate animals for the evaluation of the primary irritant potential and the maximum subirritant concentration: 30, 10, 3 and 1% in vaseline.
Erythema reactions were observed with 10 and 30% in vaseline. 30% was, therefore, selected for the epidermal induction application and 3% as maximum subirritant concentration for the epidermal challenge application.

The induction was a two-stage operation. First, intradermal injections (into the neck region); second, closed patch exposure over the injection sites one week later.
First Induction, intradermal application
Three pairs of intradermal injections (0.1 mL per injection) were made simultaneously into the shaved neck of the guinea pigs as follows:
- adjuvant/saline mixture 1:1 (v/v)
- test article in sesame oil (w/v)
- test article in the adjuvant saline mixture (w/v)
Second Induction, epidermal application
One week later the test item was incorporated in vaseline (w/w) and applied on a filterpaper patch to the neck of the animals (patch 2x4 cm; approx. 0.4 g paste per patch; occluded administration for 48 hours).

Challenge
Two weeks after the epidermal induction application the animals were tested on the flank with test material in vaseline (w/w) and the vehicle alone (patch 2x2 cm; approx. 0.2 g paste per patch; occluded administration for 24 hours).

Twenty four hours after removing the dressings, the challenge reactions were graded according to the Draize scoring scale. A second evaluation was made 48 hours after removing the dressings. The sensitising potential of the test item was classified according to the grading of Magnusson and Kligman. The body weight was recorded at start and end of the test.

Observations and records
Induction reactions
After the intradermal and the epidermal induction application irritant reactions are normally induced by the adjuvant, the high test article concentration, or the sodium lauryl sulfate pretreatment. Because most of the reactions are treatment related and not compound related, the reactions are only described in special cases in the section of results.
Challenge reactions
Twenty four hours after removing the dressings, the challenge reactions were graded according to the Draize scoring scale. A second evaluation was made 48 hours after removing the dressings.

The sensitising potential of the test item was classified according to the grading of Magnusson and Kligman. The body weight was recorded at start and end of the test.

Challenge controls:

A control group was treated with adjuvant and the vehicle during the induction period. During the challenge period the group was treated with the vehicle as well as with the test article (at least 10 animals) to check the maximum subirritant concentration of the test article in adjuvant treated animals.

Positive control substance(s):

no

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

3%

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

none

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

3%

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

none

Interpretation of results:

GHS criteria not met

Conclusions:

In the GPMT, none of the guinea pigs of the test group showed skin reactions 24 and 48 hours after challenge with 3% test item.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The skin sensitizing potential of the test article was assessed using the radioactive Murine Local Lymph Node Assay. Groups of 5 female CBA/CaOlaHsd mice each were treated with 10%, 25% and 50% (w/w) preparations of the test substance in MEK (methyl ethyl ketone) or with the vehicle alone. Each test animal was treated with 25 μL per ear of the appropriate test-substance preparation applied to the dorsal surfaces of both ears on three consecutive days. The control group was treated with 25 μL per ear of the vehicle alone. Three days after the last application, 20 μCi ³H-thymidine in 250 μL sterile saline were injected into the tail vein of the mice. About 5 to 6 hours after the ³H-thymidine injection, the mice were sacrificed and the auricular lymph nodes were removed. Lymph node response was evaluated measuring ³H-thymidine incorporation. Cell counts and weights of each animal’s pooled lymph nodes were also determined. In addition, a 0.8 cm diameter sample was punched out of the apical part of each ear, and for each animal, the weight of the pooled punches was determined to obtain an indication of possible skin irritation. No signs of systemic toxicity were noticed in all animals during general observation. When applied as 25% and 50% preparation in MEK, the test substance induced a biologically relevant (increase to 3-fold or above of control value = stimulation index (SI) ≥ 3), statistically significant and concentration-dependent increase of 3H-thymidine incorporation into the auricular lymph node cells. Concomitantly, the 25% and 50% test-substance preparation induced a biologically relevant, statistically significant and concentration-dependent response (increase to 1.5-fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts. In addition, statistically significant, biologically relevant and concentration-dependent increase in lymph node weights was observed at the 25% and 50% concentration. The increase at the 10% concentration was also statisticially significant. The test-substance concentrations did not cause increases (SI ≥ 1.25) in ear weights demonstrating the absence of relevant ear skin irritation. The increases at the 25% and 50% test-substance preprations were statistically significant. Thus, it is concluded that the test item exhibits a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen. The threshold concentration for sensitization induction was >10% <25%. The EC 3 (estimated concentration that leads to the SI of 3.0) for 3H-thymidine incorporation and the EC 1.5 (estimated concentration that leads to the SI of 1.5) for cell count was calculated by linear regression from the results of these concentrations to be 18.8% and 18.4%, respectively.

Assessment of structural analogue substance (EC 406-040-9)

The assessment of skin sensitization was performed by two guinea pig maximization studies (Ciba-Geigy, 1987 and Ciba-Geigy, 1989). Both studies were performed following OECD testing guideline 406 and under GLP. In the first study, 10% were used as the highest concentration for challenge whereas for the second study, 3% was used. In both cases, none of the 20 animals in the test group showed skin reactions. In the first study, the following concentrations were examined on separate animals for the evaluation of the primary irritant potential and the maximum subirritant concentration: 30 and 10 % in vaseline. Erythema reactions were observed with 30% in vaseline. 30% was, therefore, selected for the epidermal induction application and 10% as maximum subirritant concentration for the epidermal challenge application.

Respiratory sensitisation

Endpoint conclusion

Additional information:

No experimental data is available regarding respiratory sensitization. As the substance is a viscous liquid of low volatility, inhalation route of exposure is not considered relevant.

Justification for classification or non-classification

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

Contradicticng data are currently available, further studies are ongoing to address this issue. For now, no classification is warranted.