Benzyl cinnamate

Endpoint summary

Description of key information

The substance is a skin sensitiser Category 1B.

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:

29.07 - 11.08.2004

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:

(2002)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

(2003)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/Ca/Ola/Hsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Age at study initiation: 8-12 weeks
- Housing: maximum 4 mice per cage
- Diet: ad libitum RM1 supplied by Specail Diet Services Limited, Witham, Essex, UK
- Water: ad libitum (automatic system)
- Acclimation period: at least 5 days
- Enrichment: tents, bases and nestlets

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 3°C
- Humidity: 30-70%
- Air changes: a minimum of 15 changes per hour
- Photoperiod (hrs dark / hrs light): artifical: 12 hours light, 12 hours dark

JUSTIFICATION FOR TEST SYSTEM SELECTION: CBA/Ca mouse was chosen because previous examination of strain difference in lymphocyte proliferation responses to skin sensitisers showed this mouse strain to exhibit the most vigorous response

Vehicle:

other: 1:3 ethanol:diethyl phthalate (1:3 EtOH:DEP)

Concentration:

2.5; 5; 10; 25 or 50% w/v

No. of animals per dose:

4

Details on study design:

Dose preparation:
- within 24 hours after preparation the doses were used
Test substance
- doses: 25µL of 2.5; 5; 10; 25 or 50% w/v test item in 1:3 EtOH:DEP
- dorsal surface: ear

Vehicle control group: 1:3 EtOH:DEP alone

Procedure:
- daily for 3 consecutive days
- 3 days after last application: injection of 250 µL of phosphate buffered saline (PBS) + 20 µCi of 2.0 Ci/mmol specific activity 3H-methyl thymidine
- 5 hours later animals were killed (inhalation of halothane vapour + cervical dislocation)
- draining auricular lymph nodes were removed and placed in PBS
- preparation of single cell suspension
- cell suspensions were washed and 5% w/v trichloroacetic acid (TCA) was added
- after overnight precipitation at 4°C the pellets obtained by centrifugation were resuspended in TCA and transferred to scintillation vials
- scintillant was added prior to beta-scintillation counting (Packard Tri-Carb Liquid Scintillation Counter)

Clinical observations: daily for signs of systemic toxicity

Body weights: prior to dosing on day 1 and prior to injection on day 6.

Positive control study:
- sensitisation potential of hexylcinnamaldehyde was assessed using the procedure described above
- 25µL of 5; 10 or 25% w/v of hexylcinnamaldehyde in 4:1 acetone:olive oil
- vehicle control group: 4:1 acetone:olive oil alone

Positive control substance(s):

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

Positive control results:

5, 10 and 25% w/v in 4:1 acetone:olive oil resulted in a greater than 3-fold increase in isotope incorporation at both 10 and 25% concentrations.

Key result

Parameter:

EC3

Value:

18.4

Skin sensitisation potential of the test item:

Conc. of test subst. (% w/v)	# lymph nodes assayed	Disintegrations per minute (dpm)	Disintegrations per lymph node	Test:control ratio
0 (vehicle)	8	3254	407	n/a
2.5	8	2546	318	0.8
5	8	6255	782	1.9
10	8	6798	850	2.1
25	8	11875	1484	3.7
50	8	19113	2389	5.9

 

Interpretation of results:

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

Conclusions:

The test substance diluted in vehicle 1:3 EtOH: DEP is a skin sensitiser under the conditions of the test. The EC3 value was calculated to be 18.4 % w/v (4600 µg/cm2)

Executive summary:

In the current study the skin sensitisation potential of the test substance was assessed using the local lymph node assay. The estimated concentration giving rise to a 3-fold increase in lymphocyte proliferation was calculated. The study was performed according to OECD Guideline 429 and GLP.

The test substance was applied as 2.5, 5, 10, 25 or 50% w/v preparations in 1:3 ethanol:diethyl phthalate (1:3 EtOH:DEP). A vehicle control group was similarly treated using 1:3 EtOH:DEP alone. The estimated concentration giving rise to a 3-fold increase in lymphocyte proliferation (EC3) was calculated as percentage dose and µg/cm².

The test substance had the capacity to cause skin sensitisation when applied as 25 and 50% w/v preparations in 1:3 EtOH:DEP. The EC3 value was estimated to be 18.4% w/v (4600 µg/cm²).

The positive control, hexylcinnamaldehyde, showed the capacity to cause skin sensitisation when applied as 10 or 25% w/v preparations in 4:1 acetone:olive oil, confirming the validity of the protocol used for this study.

According to the Tables 3.4.3 and 3.4.4 of the CLP Regualtion No 1272/2008 the test item is to be classified as a skin sensitiser category 1B because the EC value > 2%.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

For the skin sensitisation endpoint there are 3 studies available. The result of the key study (2005) is used for classification purposes, while the results of the supporting studies are indicative. Nevertheless, taken together it is evident that the substance is a skin sensitiser.

In the key study (2005) the skin sensitisation potential was assessed in a local lymph node assay according to OECD Guideline 429 and GLP. The doses were 2.5, 5, 10, 25 or 50% w/v in 1:3 ethanol:diethyl phthalate (1:3 EtOH:DEP). The estimated concentration giving rise to a 3-fold increase in lymphocyte proliferation (EC3) was calculated to be 18.4% w/v (4600 µg/cm²) and the test substance had skin sensitisation effects at doses of 25 and 50% w/v.

All supporting studies were not GLP and no specific OECD guideline was followed.

In the first supporting study (1988) a modified Freund's Complete Adjuvant (FCA) test with guinea pigs was used to assess the sensitisation effects of the test substance. The concentrations used were 3 and 10%. 20% of the animals had positive reactions with 3% of the test item, while 40% of the animals showed positive reactions with 10% test item.

In the second supporting study (1977) the sensitising effects of the substance were assessed in an Open Epicutaneous Test (OET) in guinea pigs. The minimum sensitising concentration was determined to be 3% and the minimum eliciting concentration was 0.3%.

In conclusion, most supporting studies confirmed the observation of the key study and therefore, it can be concluded that the substance is a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin sensitisation

The key study (2005) resulted in an EC3 value of 18.4 %. According to the Tables 3.4.3 and 3.4.4 of the CLP Regulation No 1272/2008 a substance is to be classified as a skin sensitiser category 1B when the EC value > 2%, and category 1A when the EC value is =< 2%. Therefore, the test item is estimated to be a category 1B skin sensitiser.