Vanillin

Endpoint summary

Description of key information

A dermal sensitization study performed in adult Dunkin-Hartley guinea pigs and using the method of Maximization (Hazleton, 1992 - reliability 2) has been selected as a key study.  The macroscopic examinations did not reveal any lesion of delayed hypersensitivity in the 20 treated animals.
From the result obtained under the experimental conditions, vanillin did not provoke any reaction of cutaneous sensitization in the animals examined.

These results are confirmed by 3 other reliable experimentale studies and by human studies.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 06-JUN-1991 to 17-FEB-1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to a recognised test guideline, and in compliance with GLP. The purity of the test substance in unknown.

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

Cited as Directive 84/449/EEC, B.6

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Study performed before the adoption of the LLNA OECD guideline

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River France
- Age at study initiation: young adult
- Weight at study initiation: 300 - 500 g
- Housing: 5 per cage (same sex), in polystryrene cages
- Diet: UAR formule 114, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least one week

ENVIRONMENTAL CONDITIONS
- Temperature: 20 +/- 3 °C (a maximum was noted at 31°C)
- Humidity: 18 - 81 %
- Air changes: data not available
- Photoperiod: 12 hrs light, 12 hrs dark

IN-LIFE DATES: from 30-JUL-1991 to /

Route:

intradermal and epicutaneous

Vehicle:

other: ethanol

Concentration / amount:

35% (intradermal induction), 73% (epicutaneous induction and challenge)

Route:

epicutaneous, occlusive

Vehicle:

other: ethanol

Concentration / amount:

35% (intradermal induction), 73% (epicutaneous induction and challenge)

No. of animals per dose:

10 males and 10 females

Details on study design:

*** RANGE FINDING TEST:
Subcutaneous injection of vanillin in a 35% solution provoked a moderate irritation with burnt aspect during the preliminary study.
The epicutaneous application of a 73% (w/w) paste in absolute ethanol provoked a weak irritation during the preliminary study.

*** MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2
- type of epicutaneous induction: occlusive
- SLS application: yes (at 10% in Codex paraffin)
- Exposure period: 48 hrs (epicutaneous)
- Test groups: test substance in FCA
- Control group: FCA only
- Site: back
- Frequency of applications: on days 1 and 9
- Duration: 9 days
- Concentrations: 35% (intradermal induction), 73% (epicutaneous induction)

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: day 22
- Exposure period: 24 hrs
- Test groups: Test substance
- Control group: Test substance
- Site: back
- Concentrations: 73%
- Evaluation (hr after challenge): 48 and 72 hrs

Challenge controls:

no data

Positive control substance(s):

no

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

73%

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

none

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

73%

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

none

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

73%

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

None

Reading:

2nd reading

Hours after challenge:

72

Group:

negative control

Dose level:

73%

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

None

Reading:

1st reading

Hours after challenge:

48

Group:

positive control

Remarks on result:

not measured/tested

Reading:

2nd reading

Hours after challenge:

72

Group:

positive control

Remarks on result:

not measured/tested

RESULT OF THE TEST:

Reading	Hours after challenge	Group	No. of animals with (+) reactions
1st reading	48	test group	0/20
2nd reading	72	test group	0/20
1st reading	48	control group	0/20
2nd reading	72	control group	0/20

Interpretation of results:

GHS criteria not met

Executive summary:

In a dermal sensitization study (Hazleton, 1992) with Vanilline cristallisée (purity unknown) in ethanol, young adult Dunkin-Hartley guinea pigs (10/sex) were tested using the method of Maximization.
Signs of irritation were noted during the induction.
The macroscopic examinations did not reveal any lesion of delayed hypersensitivity in the 20 treated animals. No cutaneous abnormality was noted in the 20 guinea pigs examined in the control group.
From the result obtained under the experimental conditions employed, vanillin did not provoke any reaction of cutaneous sensitization in the animals examined.

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The test conditions are well described and are in accordance with the recognised EC and OECD test guidelines. The GLP are not mentioned.

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

Clinical signs and body weight were not observed.

GLP compliance:

not specified

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Olac, Bicester, Oxfordshire, UK
- Age at study initiation: 6 to 12 weeks old
- Weight at study initiation: no data
- Housing: under standard conditions, no more data.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS: no data

IN-LIFE DATES: no data

Vehicle:

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

Concentration:

0, 2.5, 5, 10, 25 and 50%

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS: no data

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: no data
- Criteria used to consider a positive response: The test item should be regarded as a skin sensitizer when the SI for a dose group is >=3. Other relevant criteria such as cellularity, radioactivity levels and ear thickness will be also taken into account to evaluate the data.
When appropriate, the EC3 value (theoretical concentration resulting in a SI value of 3) will be determined by linear interpolation of points on the dose-response curve, immediately above and below the 3-fold threshold. The equation used for calculation of EC3 will be:
EC3 = c + [(3 - d)/(b - d)] x (a - c)
Where a = the lowest concentration giving stimulation > 3; b = the actual stimulation index caused by a; c = the highest concentration failing to produce a stimulation index of 3; and d = the actual stimulation index caused by c.

TREATMENT PREPARATION AND ADMINISTRATION: Groups of mice were exposed topically on the dorsum of both ears to 25 µl of various concentrations of Vanillin, or to the same volume of vehicle Acetone/ olive oil (4/1) alone, daily for 3 consecutive days. 5 days after the initiation of exposure, all mice were injected intravenously via the tail vein with 25 µCi of[H]methyl thymidine in 250 µl of phosphate buffered saline (PBS).
5 hours later, mice were killed and the draining auricular lymph nodes were excised and pooled for each experimental group.

Statistics:

no

Parameter:

SI

Remarks on result:

other: see the following Table 1

Table 1: Stimulation Index

Test or Control Group	0	2,5%	5%	10%	25%	50%
Stimulation Index (SI)	1	0,87	1,42	1,48	1,23	1,39

Interpretation of results:

GHS criteria not met

Conclusions:

Classification: not sensitizing

Executive summary:

In a dermal sensitization study (Basketter et al., 2001) with Vanillin in acetone/olive oil (4:1 v/v), CBA/Ca mouse (4 female) were testing using the method of Local Lymph Node Assay (LLNA). During the study no positive lymphoproliferative response were observed.

In this study, Vanillin is not a dermal sensitizer.

Reference 3

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study well described. The guideline for Buehler test was modified and scientifically questionable for the other substances than Vanillin tested. Some details on environmental conditions are missing. Contradictory informations were indicated in table of results and text in publication. The purity of tested vanillin is unknown.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

not specified

GLP compliance:

not specified

Type of study:

other: Modified Buehler test and guinea pig Patch test

Species:

guinea pig

Strain:

other: albino

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source:
- Age at study initiation: 6-8 weeks old
- Weight at study initiation: 300 to 450 g
- Housing: in separate cages
- Diet: animals were fed on a pellet diet, green vegetables
- Water: standard tap water ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS: no data

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

pure product (100%)

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

pure product (100%)

No. of animals per dose:

5 or 10

Details on study design:

***Patch test on guinea pigs:
- 5 animals used to determined the treshold irritation concentration
- Exposure period: 48 hours
- Test group: 5% and 100%
- Control group: white petrolatum
- site: flank
- frequency: one
- Readings: 1, 24 and 48 hours after removal of the material.
- Amount applied: 0.1 g

*** Modified Buehler test.
MAIN STUDY
A. INDUCTION EXPOSURE (10 animals)
- Exposure application: 24 hours
- Test groups: 1 treated with pure product (10 animals)
- Control group: 1 treated with petrolatum (10 animals)
- Site: nape
- Frequency of applications: 3 times a weeks for 2 weeks.
- Duration: 24 hours
- Concentrations: 10%

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: Challenge was done 2 weeks after final induction.
- Exposure period: 48 hours
- Test groups: 1 treated with pure product (10 animals)
- Control group: 1 treated with petrolatum (10 animals)
- Site: left flank
- Concentrations: pure product
- Evaluation (hr after challenge): 1, 24 and 48 hours after removal of the substance.

***Cross reactivity
- Sensitized guinea pigs were re-challenged for evaluation of cross reactivity 7 days after challenge with pure Vanillin.
- Site: right flank

REMARK: the Vanillin was studied with other compounds of Vanillin process: DMA (10%), SN (2%), p-NDMAH (1%) for patch test and 10% for Buehler induction, and 2% and 5% for challenge test and different concentrations for cross-reactivity.

Challenge controls:

10 animals treated with pure petrolatum

Positive control substance(s):

no

Reading:

other: Challenge

Hours after challenge:

48

Group:

test chemical

Dose level:

pure product

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

other: Reading: other: Challenge. . Hours after challenge: 48.0. Group: test group. Dose level: pure product. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.

Reading:

other: Challenge

Hours after challenge:

48

Group:

negative control

Dose level:

pure product

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

other: Reading: other: Challenge. . Hours after challenge: 48.0. Group: negative control. Dose level: pure product. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.

Reading:

other: Patch test

Hours after challenge:

48

Group:

test chemical

Dose level:

100%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

none

Remarks on result:

other: Reading: other: Patch test. . Hours after challenge: 48.0. Group: test group. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: none.

Reading:

other: Cross-reactivity

Hours after challenge:

168

Group:

test chemical

Dose level:

100%

No. with + reactions:

0

Clinical observations:

no data

Remarks on result:

other: Reading: other: Cross-reactivity. . Hours after challenge: 168.0. Group: test group. Dose level: 100%. No with. + reactions: 0.0. Clinical observations: no data.

Interpretation of results:

GHS criteria not met

Conclusions:

Pure Vanillin was negative in a modified Buehler test, and Patch test on guinea pigs.

Executive summary:

A study (Wang et al, 1987) investigated the skin irritation and sensitizing potential of chemicals in manufacture of Vanillin, using specific process with urotropine.

In this study, male and female albino guinea pig were tested with Vanillin at 10 or 100% using a modified Buehler test and at 100 % using a patch test.

No signs of irritation were noted.

In Buehler test the induction was done by topical application (occlusive) at 10% with Vanillin in petrolatum for 24 -hour application, 3 times in 2 weeks on napes of 10 animals.

The challenge was done 2 weeks after final induction by topical application (occlusive) with 100% Vanillin on left flanks.

No signs of sensitization were observed.

A cross-reactivity was done with other substances and did not revealed cross-sensitization.

Based on these results, Vanillin was neither irritant nor sensitizer.

Reference 4

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study well described. The authors considered results of Vanillin as "false negative" with no argument. As no argument was given, results of this study were not taken into account and result "negative" were considered valid. No other information that the own interpretation of the authors could permit to consider that Vanillin is a "false negative".

Qualifier:

no guideline followed

GLP compliance:

not specified

Type of study:

mouse ear swelling test

Species:

mouse

Strain:

CF-1

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Age at study initiation: 6 to 8 weeks old
- Acclimation period: 1 week
No more data

Route:

epicutaneous, open

Vehicle:

other: ethanol 70%

Concentration / amount:

50% in ethanol

Route:

epicutaneous, open

Vehicle:

other: ethanol 70%

Concentration / amount:

50% in ethanol

No. of animals per dose:

10 or 15 animals in test groups
5 or 10 animlas in control group

Details on study design:

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 4 (Days 0, 1, 2 and3)
- Exposure period: 7 days
- Control group: yes, solvent alone
- Site: topically on the stomach region
- Frequency of applications: daily for 3 concutive days
- Duration: 7 days
- Concentrations: 50% in ethanol

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 10
- Exposure period: 24 and 48 hours
- Control group: yes
- Site: left ear of each animal
- Concentrations: 50% in ethanol
- Evaluation (hr after challenge): 24 and 48 hours

All mice were topically dose with 100 µl of test material in solvent or solvent alone (controls) applied to the center of the shaved region. The application was allowed to dry before the animal was returned to its cage. Tape stripping and topical application of the appropriate solution to the stomach were repeated for 3 additional consecutive days.
Seven days after the final topical application to the stomach, 20 µl of the test compound in solution was applied to the left ear of each animal (test and control) and 20 µl of the solvent was applied to the right ear.
At both 24 an 48 hours after this challenge, animals were lightly anesthetized with ether and the thicknesses of both ears were measured.
The percentage of ear swelling determine the sensitisation effect.

EVALUATION CRITERIA:
Positive responses were defined on an animal-by-animal basis as cases where the test ear demonstated at least a 20% greater thickness than the control ear (it being determined that variations in ear thickness and occasional low-level irritation responses always gave increase no greater than 10% and that responses between 10 and 20% occured at a frequency of approximately 0.5%). With this incidence, percentage responsiveness was calculated. The probability of a false positive due to variations in ear thickness was less than 0.1%.
%ear swelling = [(test ear thickness - control ear thickness)/control ear thickness] * 100

Reading:

other: no data

Hours after challenge:

24

Group:

test chemical

Dose level:

50%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

other: Reading: other: no data. . Hours after challenge: 24.0. Group: test group. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.

In this test, percentage of ear swelling was 4% for vanillin. This result is below the level of 20% for positive results. 0% of animals were sensitized.

Vanillin was negative in Mouse Ear Swelling Test (MEST).

Interpretation of results:

GHS criteria not met

Conclusions:

Vanillin was negative in MEST.

Executive summary:

In a study (Gad et al, 1986), CF-1 female mice were tested in a Mouse Ear Swelling Test (MEST with Vanillin (purity unknown).

All mice were topically dose with 100 µl of Vanillin in solvent (50%) or solvent alone (controls) applied to the center of the shaved region. The application was allowed to dry before the animal was returned to its cage. Tape stripping and topical application of the appropriate solution to the stomach were repeated for 3 additional consecutive days.

Seven days after the final topical application to the stomach, 20 µl of Vanillin in solution (50%) was applied to the left ear of each animal (test and control) and 20 µl of the solvent was applied to the right ear.

At both 24 an 48 hours after this challenge, animals were lightly anesthetized with ether and the thicknesses of both ears were measured.

Vanillin presented a percentage of swelling of 4%. In these conditions Vanillin could be considered as negative in MEST, and Vanillin was not sensitizing in this test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Several animal studies were available for skin sensitization with Vanillin. Amongs of them, four were of reliability 2; One of them was selected as key study and had reliability 2 (Hazleton, 1992), and the others were selected as supporting studies with reliability 2 (Basketter, 2001; Gad, 1986; Wang, 1987).

The key study was a maximization test, and the summary is the following:

In a dermal sensitization study (Hazleton, 1992) with Vanilline cristallisée (purity unknown) in ethanol, young adult Dunkin-Hartley guinea pigs (10/sex) were tested using the method of Maximization. The doses tested for induction and challenge phases were: 35% (intradermal induction), 73% (epicutaneous induction and challenge)
Signs of irritation were noted during the induction.
The macroscopic examinations did not reveal any lesion of delayed hypersensitivity in the 20 treated animals. No cutaneous abnormality was noted in the 20 guinea pigs examined in the control group.
From the result obtained under the experimental conditions employed, vanillin did not provoke any reaction of cutaneous sensitization in the animals examined.

Two other studies were a LLNA and a MEST tests. In the first one (Basketter et al., 2001), CBA/Ca mice (4 females) were testing with Vanillin in acetone/olive oil (4:1 v/v), using the method of Local Lymph Node Assay (LLNA) at doses of 0, 2.5, 5, 10, 25 and 50%. During the study no positive lymphoproliferative response were observed at all doses tested. In this study, Vanillin was not a dermal sensitizer.

In the study of Gad et al (1986) CF-1 female mice were tested in a Mouse Ear Swelling Test (MEST) with Vanillin (purity unknown).All mice were topically dose with 100 µl of Vanillin in solvent (50%) or solvent alone (controls) applied to the center of the shaved region. The application was allowed to dry before the animal was returned to its cage. Tape stripping and topical application of the appropriate solution to the stomach were repeated for 3 additional consecutive days. Seven days after the final topical application to the stomach, 20 µl of Vanillin in solution (50%) was applied to the left ear of each animal (test and control) and 20 µl of the solvent was applied to the right ear. At both 24 an 48 hours after this challenge, animals were lightly anesthetized with ether and the thicknesses of both ears were measured. Vanillin presented a percentage of swelling of 4%. In these conditions Vanillin could be considered as negative in MEST, and Vanillin was not sensitizing in this test.

The study of Wang et al (1987) investigated the skin irritation and sensitizing potential of chemicals in manufacture of Vanillin, using specific process with urotropine. In this study, male and female albino guinea pig were tested with Vanillinat 10or 100% using a modified Buehler test and at 100 % using a patch test. No signs of irritation were noted. In Buehler test the induction was done by topical application (occlusive) at 10% with Vanillin in petrolatum for 24 -hour application, 3 times in 2 weeks on napes of 10 animals. The challenge was done 2 weeks after final induction by topical application (occlusive) with 100% Vanillin on left flanks. No signs of sensitization were observed. A cross-reactivity was done with other substances and did not revealed cross-sensitization. Based on these results, Vanillin was neither irritant nor sensitizer.

Five other studies were not taken into account, because they had reliability 3 or 4, due to lack of details of the study, to permit to consider them for the assessment.

Three Human data with validity 2 confirmed the results obtained in animal studies. These studies were Patch tests on Humans (Hausen, 2001; Francalanci, 2000; Wang, 1987).

In the study of Hausen (2001), occlusive patch of 5% of Vanillin were applied for 24h to 102 volunteers already positive to BalsamPeru. Vanillin gave negative results on patients already sensitized with Balsam of Peru.

In the study of Francalanci (2000), 54 patients who presented eczematous lesions on the lips probably due to toothpastes, received occlusive patch test with 10% of Vanillin for two days. 1/54 patient react positively to Vanillin, it is not sufficient to consider Vanillin as sensitizing.

In the study of Wang (1987), Vanillin was tested with other substance of urotropine process for the manufacturing of the Vanillin. 0/45 patients had positive results, so Vanillin was not sensitizing in this patch test study

See section 7.10.4 for more details.

All the results available indicated that Vanillin was not a skin sensitizer. According to classification criteria of EC regulation 1272/2008 Vanillin should not be classified for sensitisation.

Vanilline is not sensitizing to skin based on studies on animals and humans.

Justification for classification or non-classification

Vanillin is not a skin sensitizer based on studies on animals and humans. Therefore no classification is required according to CLP criteria.