Benzyl benzoate

Endpoint summary

Description of key information

A guideline and GLP-compliant LLNA is available for benzyl benzoate.

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:

21 to 27 July 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline and GLP-compliants study

Qualifier:

according to guideline

Guideline:

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

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
- Source: Harlan Interfauna UK limited, Bicester, UK
- Age at study initiation: 8-12 weeks
- Weight at study initiation: circa 16-20g
- Housing: 4 mice per cage
- Diet (e.g. ad libitum): RM1 from Special Diet Services, ad libitum
- Water (e.g. ad libitum): mains water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 30-70
- Air changes (per hr):15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 21 July 2004 To: 27 July 2004

Vehicle:

other: 1:3 ethanol:diethylphthalate (1:3 EtOH:DEP)

Concentration:

2.5, 5, 10, 25 or 50% v/v

No. of animals per dose:

Four female mice

Details on study design:

RANGE FINDING TESTS:
Not performed

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local lymph node assay according to methods of Kimber et al.
- Criteria used to consider a positive response: Stimulaton Index SI value of 3.0 or greater and EC3 value where this can be calculated

TREATMENT PREPARATION AND ADMINISTRATION:
Groups of four female mice were allocated to the study and approximately 25 µL of a 2.5, 5, 10, 25 or 50% v/v preparation of benzyl benzoate in 1:3 EtOH:DEP was applied to the dorsal surface of each ear. A vehicle control group was similarly treated.
Treatment was repeated on three consecutive days and then three days later all animals were intravenously injected, via the tail vein, with 250µL of phosphate buffered saline containing 20 µCi of a 2.0 µCi/mmol specific activity 3H-methyl thymidine. The mice were killed 5 hours later and draining auricular lymph nodes were excised.

Single cell suspensions were prepared in PBS and subject to cycles of centrifugation and following addition of scintillation fluid, the lymph node suspensions were counted using a ß-scintillation counter.

Positive control substance(s):

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

Statistics:

Stimulation Indices evaluated using simple t-test.
EC3 value calculated by interpolation on the SI axis

Positive control results:

Hexylcinnamaldehyde concentrations of 10 and 25% w/v in acetone:olive oil caused an increase in isotope incorporation with SI values of greater than 3 in each case, confirming the validity of the assay methods.

Parameter:

SI

Value:

0.7

Test group / Remarks:

Benzyl benzoate concentration: 2.5%

Parameter:

SI

Value:

0.7

Test group / Remarks:

Benzyl benzoate concentration: 5%

Parameter:

SI

Value:

0.8

Test group / Remarks:

Benzyl benzoate concentration: 10%

Parameter:

SI

Value:

1.7

Test group / Remarks:

Benzyl benzoate concentration: 25%

Parameter:

SI

Value:

2.7

Test group / Remarks:

Benzyl benzoate concentration: 50%

Parameter:

other: DMP (disintegrations per minute)

Value:

2 248

Test group / Remarks:

0 (vehicle control)

Remarks on result:

other: SI = NA

Parameter:

other: DMP (disintegrations per minute)

Value:

1 538

Test group / Remarks:

Benzyl benzoate concentration: 2.5%

Remarks on result:

other: SI = 0.7

Parameter:

other: DMP (disintegrations per minute)

Value:

1 662

Test group / Remarks:

Benzyl benzoate concentration: 5%

Remarks on result:

other: SI = 0.7

Parameter:

other: DMP (disintegtations per minute)

Value:

1 843

Test group / Remarks:

Benzyl benzoate concentration: 10%

Remarks on result:

other: SI = 0.8

Parameter:

other: DMP (disintegrations per minute)

Value:

3 798

Test group / Remarks:

Benzyl benzoate concentration: 25%

Remarks on result:

other: SI = 1.7

Parameter:

other: DMP (Disintegrations per minute)

Value:

6 024

Test group / Remarks:

Benzyl benzoate concentration: 50%

Remarks on result:

other: SI = 2.7

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

The increase in disintegrations per minute or per lymph node was less than threefold for all tested concentrations. An EC3 was not calculated but was estimated to exceed 50% (greater than the maximum administered dose of 12500 µg/cm2). SI values ranged from 0.7 to 2.7, generally increased in a linear manner with increasing dose, but none exceeded the threshold value of 3.0

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Refer to table below. DPM were in range of 1538 to 6024 for 2.5 to 50% concentrations and the control group was 2248.

 

Benzyl benzoate concentration (% w/v)	Number of lymph nodes assayed	Disintegrations per minute (DPM)	DPM per lymph node	Test : control ratio (Stimulation Index, SI)
0 vehicle control	8	2248	281	NA
2.5	8	1538	192	0.7
5	8	1662	208	0.7
10	8	1843	230	0.8
25	8	3798	475	1.7
50	8	6024	753	2.7
EC3	Estimated to exceed 50% or 12500 µg/cm^2

 

Interpretation of results:

GHS criteria not met

Conclusions:

None of the calculated SI values exceeded the classification threshold of 3.0 and no EC3 value could be calculated from the pattern of responses.

Executive summary:

A local lymph node assay was completed using standard procedures but with additional treatment groups. Groups of four female mice were dosed topically at various concentrations on three consecutive days and then injected with tritiated thymidine, following a period of time to allow for lymphocyte proliferation and incorporation of the labelled thymidine, the lymph nodes were excised and prepared or scintillation counting.

The level of isotope incorporation was less than 3 -fold that of the control for treatment group and consequently benzyl benzoate is not considered to be a potential sensitiser.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

A local lymph node assay was completed using standard procedures but with additional treatment groups. Groups of four female mice were dosed topically at various concentrations on three consecutive days and then injected with tritiated thymidine, following a period of time to allow for lymphocyte proliferation and incorporation of the labelled thymidine, the lymph nodes were excised and prepared or scintillation counting. The level of isotope incorporation was less than 3 -fold that of the control for treatment group and consequently benzyl benzoate is not considered potential sensitiser.

Justification for selection of skin sensitisation endpoint:
Only one study available for this endpoint

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.