4-methoxybenzyl alcohol

Endpoint summary

Description of key information

Challenge patch with the highest non irritant solution in animals pre-treated with the preparation 60% and 30% are positive in half of the animals.The test chemical can be considered to cause sensitzing reactions to guinea pigs in the OET. The test chemical was considered to be one of the weak contact sensitiser for this species.

The relative potency index of the test chemical was calculated to be 5.9.

Based on the relative potency index, the test chemical was considered to be a moderate sensitizer.

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

data is from experimental reports

Qualifier:

according to guideline

Guideline:

other: Open Epicutaneous test

Principles of method if other than guideline:

Open Epicutaneous test was carried out to evaluate the dermal sensitization potential of the test chemical

GLP compliance:

not specified

Type of study:

open epicutaneous test

Justification for non-LLNA method:

not specified

Species:

guinea pig

Strain:

not specified

Sex:

not specified

Route:

epicutaneous, open

Vehicle:

other: acetone

Concentration / amount:

60, 30, 10 and 3% solutions

Adequacy of induction:

not specified

No.:

#1

Route:

epicutaneous, open

Vehicle:

other: acetone

Concentration / amount:

60, 30, 10 and 3% solutions

Adequacy of challenge:

not specified

No. of animals per dose:

6-8

Details on study design:

Induction:
Days 0-20: On day 0, 0.1ml of the test material, undiluted and, if possible or necessary of its progressively diluted solutions or emulsions or suspensions is applied to an area measuring 8cm2 on the clipped flank skin of 6-8 guinea pigs per concentration group, using 1-6 such groups for each test material.
The applications are repeated daily for 3 weeks or done 5 times weekly during 4 weeks, always using the same skin site. The application site is left uncovered and the reactions, if continuous daily applications are performed, can be read 24 hours after each application or at the end of each week. When very strong skin reactions are provoked, the application site is changed.
Challenge:
Day 21 &35: to determine whether or not contact sensitization was induced, all groups of guinea pigs previously treated for 21 days as described above, as well as 6-8 untreated or only with the vehicle pretreated controls are tested on days 21 and 35 on the contralateral flank with the test material at the minimal irritating and some lower concentrations. The minimal irritating concentration of each material is used in order to confirm the biological activity determined before starting the induction (day-1) and to exclude false results based on instability of test materials. These tests are performed by applying with a pipette 0.025ml of each concentration to skin areas measuring 2 cm2, the reactions being read after 24,48 and /or 72 hours. This procedure enables to determine the minimal sensitising concentration necessary for inducing allergic contact hypersensitivity and the minimal eliciting concentration necessary to cause a positive reaction. The test material is considered allergenic at a concentration when at least one out of the 8 animals of the concentration group concerned shows positive reactions with non-irritant concentrations used for challenge, i.e its threshold concentration causing skin reactions is shifted into the lower part of the concentration range used for challenge.

Challenge controls:

6-8 untreated or only with the vehicle pretreated controls are tested on days 21 and 35 on the contralateral flank with the test material at the minimal irritating and some lower concentrations

Positive control substance(s):

not specified

Key result

Reading:

1st reading

Hours after challenge:

72

Group:

test chemical

Dose level:

60 % solution

No. with + reactions:

4

Total no. in group:

8

Clinical observations:

Challenge on day 21 and 35 of the test

Remarks on result:

positive indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

30 % solution

No. with + reactions:

4

Total no. in group:

8

Clinical observations:

Challenge on day 21 and 35 of the test

Remarks on result:

positive indication of skin sensitisation

Key result

Reading:

other: 3rd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

10 % solution

No. with + reactions:

0

Total no. in group:

8

Clinical observations:

Challenge on day 21 and 35 of the test

Remarks on result:

positive indication of skin sensitisation

Key result

Reading:

other: 4th reading

Hours after challenge:

72

Group:

test chemical

Dose level:

3 % solution

No. with + reactions:

0

Total no. in group:

8

Clinical observations:

Challenge on day 21 and 35 of the test

Remarks on result:

positive indication of skin sensitisation

Capacity to induce allergic sensitisation

After daily applications over three weeks	Sensitisation rate Number of animals (positive/ total)
Concentration %	Day 21	Day 35
60	4/8 4/8 0/8 0/8
30
10
3

Interpretation of results:

other: weak contact sensitiser

Conclusions:

Challenge patch with the highest non irritant solution in animals pre-treated with the preparation 60% and 30% are positive in half of the animals.The test chemical can be considered to cause sensitzing reactions to guinea pigs in the OET. The test chemical was considered to be one of the weak contact sensitiser for this species.

Executive summary:

Open Epicutaneous test was carried out to evaluate the dermal sensitization potential of the test chemical

On day 0 of the induction exposure, 0.1ml of the test material, undiluted and, if possible or necessary of its progressively diluted solutions or emulsions or suspensions is applied to an area measuring 8cm2 on the clipped flank skin of 6-8 guinea pigs per concentration group, using 1-6 such groups for each test material.

The applications are repeated daily for 3 weeks or done 5 times weekly during 4 weeks, always using the same skin site. The application site is left uncovered and the reactions, if continuous daily applications are performed, can be read 24 hours after each application or at the end of each week. When very strong skin reactions are provoked, the application site is changed. To determine whether or not contact sensitization was induced, all groups of guinea pigs previously treated for 21 days as described above, as well as 6-8 untreated or only with the vehicle pretreated controls are tested on days 21 and 35 on the contralateral flank with the test material at the minimal irritating and some lower concentrations. The minimal irritating concentration of each material is used in order to confirm the biological activity determined before starting the induction (day-1) and to exclude false results based on instability of test materials. These tests are performed by applying with a pipette 0.025ml of each concentration to skin areas measuring 2 cm2, the reactions being read after 24,48 and /or 72 hours. This procedure enables to determine the minimal sensitising concentration necessary for inducing allergic contact hypersensitivity and the minimal eliciting concentration necessary to cause a positive reaction. The test material is considered allergenic at a concentration when at least one out of the 8 animals of the concentration group concerned shows positive reactions with non-irritant concentrations used for challenge, i.e its threshold concentration causing  skin reactions is shifted into the lower part of the concentration range used for challenge. 6-8 untreated or only with the vehicle pretreated controls are tested on days 21 and 35 on the contralateral flank with the test material at the minimal irritating and some lower concentrations

Challenge patch with the highest non irritant solution in animals pre-treated with the preparation 60% and 30% are positive in half of the animals.The test chemical can be considered to cause sensitzing reactions to guinea pigs in the OET. The test chemical was considered to be one of the weak contact sensitiser for this species.

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

data is from peer reviewed journals

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: USEPA method OPPTS 870.2600

Principles of method if other than guideline:

To evaluate the potential of the test chemical for inducing allergic contact dermatitis

GLP compliance:

not specified

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Details on test animal
TEST ANIMALS
- Age at study initiation: 7-12 weeks

Vehicle:

other: ethanol:diethyl phthalate [1:3]

Concentration:

2,5 10, 25, and 50% w/v in 1:3 ethanol:diethyl phthalate

No. of animals per dose:

four female mice/dose

Details on study design:

Details on study design

Pre –screen tests : No data

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response:
A substance was classified as a skin sensitizer if it induced a threefold or greater increase in local lymph node proliferative activity at one or more test concentrations when compared with concurrent vehicle-treated controls (SI≥3). Dose response data were used to measure the relative skin sensitization potency of all of the chemicals that were positive. When the LLNA dose-response curve included concentrations that induced at least one SI greater than 3 and one SI less than 3, EC3 values were calculated by linear interpolation. For chemicals that induced an SI greater than or equal to 3 at all concentrations tested, an EC3 value was extrapolated from the two lowest doses used. For this extrapolation method to work, a dose response should be evident. The relative sensitizing potencies of the chemical allergens were categorized via a recently proposed arbitrary classification scheme.

TREATMENT PREPARATION AND ADMINISTRATION: groups of CBA female mice (7–12 weeks of age) were exposed topically on the dorsum of both ears to 25 µL of test material or to an equal volume of the relevant vehicle alone. Treatment was performed daily for 3 consecutive days. Five days after the initiation of exposure, all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 20 µCi of tritiated thymidine. Mice were sacrificed 5 hours later, and the draining auricular lymph nodes were excised and pooled for each experimental group or each individual animal. The incorporation of tritiated thymidine measured by beta scintillation counting was reported in disintegrations per minute (dpm). A stimulation index (SI) was calculated for each chemical-treated group as the ratio of the dpm of the treated group (or mean dpm when individual animals were assessed) to the dpm or mean dpm of the concurrent vehicle control group.

Positive control substance(s):

not specified

Key result

Parameter:

EC3

Value:

5.9

Test group / Remarks:

Moderate Sensitizer

Remarks on result:

other: positive sensitizer

Table: Chemical Structures, Molecular Weights, LLNA Data, Potency Categorizations, and Reaction Mechanistic Domains

CAS	Vehicle	LLNA%	LLNA%	LLNA%	LLNA%	LLNA%	LLNA SI	LLNA SI	LLNA SI	LLNA SI	LLNA SI	LLNA EC3	Relative Potency	Reaction mechanism domain
105-13-5	E: DEP [1:3]	2	5	10	25	50	1.8	2.8	3.9	5.1	5.3	5.9	Moderate	Pro/Pre-MA

E:D = ethanol:diethyl phthalate[1:3]; LLNA – Local lymph node assay (LLNA% = weight per volume :concentration); EC3 – Mathematically estimated concentration of the test chemical necessary to induce a threefold stimulation index; MA –Michaels acceptor

**value is estimated

Interpretation of results:

other: sensitizing

Conclusions:

The relative potency index of the test chemical was calculated to be 5.9.
Based on the relative potency index, the test chemical was considered to be a moderate sensitizer.

Executive summary:

The dermal sensitization potential ofthe test chemical was evaluated in a mouse local lymphnode assay(LLNA). The study was performed as per OECD 429 Guidelines. Groups of female CBA mice (7-12 weeks of age) were exposed topically on the dorsum of both ears to 25µl of the test material or to an equal volume of relevant vehicle only. Treatment was performed daily for 3 consecutive days.Five days after the initiation of exposure, all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 20 µCi of tritiated thymidine. Mice were sacrificed 5 hours later, and the draining auricular lymph nodes were excised and pooled for each experimental group or each individual animal. The incorporation of tritiated thymidine measured by beta scintillation counting was reported in disintegrations per minute (dpm). A stimulation index (SI) was calculated for each chemical-treated group as the ratio of the dpm of the treated group (or mean dpm when individual animals were assessed) to the dpm or mean dpm of the concurrent vehicle control group.The approach to estimation of the relative skin sensitization potential was based on the mathematical estimation of the concentration of chemical necessary to obtain a threshold positive response (SI = 3); this is termed as the EC3 value.A substance was classified as a skin sensitizer if it induced a threefold or greater increase in local lymph node proliferative activity at one or more test concentrations when compared with concurrent vehicle-treated controls (SI≥3).

The relative potency index of the test chemical was calculated to be 5.9.

Based on the relative potency index, the test chemical was considered to be a moderate sensitizer.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Various studies have been reviewed to determine the allergenic potential of the test chemical in living organisms. These include in vivo experimental studies performed on mice, guinea pigs, humans for the test chemical.

Open Epicutaneous test was carried out to evaluate the dermal sensitization potential of the test chemical.

On day 0 of the induction exposure, 0.1ml of the test material, undiluted and, if possible or necessary of its progressively diluted solutions or emulsions or suspensions is applied to an area measuring 8cm2 on the clipped flank skin of 6-8 guinea pigs per concentration group, using 1-6 such groups for each test material.

The applications are repeated daily for 3 weeks or done 5 times weekly during 4 weeks, always using the same skin site. The application site is left uncovered and the reactions, if continuous daily applications are performed, can be read 24 hours after each application or at the end of each week. When very strong skin reactions are provoked, the application site is changed. To determine whether or not contact sensitization was induced, all groups of guinea pigs previously treated for 21 days as described above, as well as 6-8 untreated or only with the vehicle pretreated controls are tested on days 21 and 35 on the contralateral flank with the test material at the minimal irritating and some lower concentrations. The minimal irritating concentration of each material is used in order to confirm the biological activity determined before starting the induction (day-1) and to exclude false results based on instability of test materials. These tests are performed by applying with a pipette 0.025ml of each concentration to skin areas measuring 2 cm2, the reactions being read after 24,48 and /or 72 hours. This procedure enables to determine the minimal sensitising concentration necessary for inducing allergic contact hypersensitivity and the minimal eliciting concentration necessary to cause a positive reaction. The test material is considered allergenic at a concentration when at least one out of the 8 animals of the concentration group concerned shows positive reactions with non-irritant concentrations used for challenge, i.e its threshold concentration causing  skin reactions is shifted into the lower part of the concentration range used for challenge. 6-8 untreated or only with the vehicle pretreated controls are tested on days 21 and 35 on the contralateral flank with the test material at the minimal irritating and some lower concentrations

Challenge patch with the highest non irritant solution in animals pre-treated with the preparation 60% and 30% are positive in half of the animals.The test chemical can be considered to cause sensitzing reactions to guinea pigs in the OET. The test chemical was considered to be one of the weak contact sensitiser for this species.

This is supported by the results of a mouse local lymphnode assay(LLNA) to evaluate the dermal sensitization potential of the test chemical. The study was performed as per OECD 429 Guidelines. Groups of female CBA mice (7-12 weeks of age) were exposed topically on the dorsum of both ears to 25µl of the test material or to an equal volume of relevant vehicle only. Treatment was performed daily for 3 consecutive days.Five days after the initiation of exposure, all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 20 µCi of tritiated thymidine. Mice were sacrificed 5 hours later, and the draining auricular lymph nodes were excised and pooled for each experimental group or each individual animal. The incorporation of tritiated thymidine measured by beta scintillation counting was reported in disintegrations per minute (dpm). A stimulation index (SI) was calculated for each chemical-treated group as the ratio of the dpm of the treated group (or mean dpm when individual animals were assessed) to the dpm or mean dpm of the concurrent vehicle control group.The approach to estimation of the relative skin sensitization potential was based on the mathematical estimation of the concentration of chemical necessary to obtain a threshold positive response (SI = 3); this is termed as the EC3 value.A substance was classified as a skin sensitizer if it induced a threefold or greater increase in local lymph node proliferative activity at one or more test concentrations when compared with concurrent vehicle-treated controls (SI≥3).

The relative potency index of the test chemical was calculated to be 5.9.

Based on the relative potency index, the test chemical was considered to be a moderate sensitizer.

These results are also supported by a Freunds Complete Adjuvant test performed to evaluate the dermal sensitization potential of test chemical.

0.05 ml of the compound (undiluted, diluted or suspended) mixed with the same volume of FCA were injected intradermally into the neck on days 0,2,4,7 and 9 (total dose approx. 250mg). The experimental animals and controls, treated with 0.05ml of FCA only, were tested epicutaneously on days 21 and 35. These tests were performed by applying 0.025ml of each test-concentration to skin areas measuring 2 sq.cm. The reactions were read after 24, 48 and 72 hours.

The test chemical showed signs of sensitization in all the guinea pigs tested on day 21 and day 35 of the challenge exposure.

Hence, the test chemical can be considered to be sensitizing to guinea pig skin.

Draize test was performed to determine the dermal sensitization potential of the test chemical in guinea pigs.

On day 0, groups of 8 animals were injected intradermally with 0.05ml of a 0.1% solution of the preparation in saline and with 0.1 ml on nine further alternate days (total dose: 0.95mg). Experimental and control animals were tested intradermally with 0.05ml of a 0.1% solution on days 35 and 49, the mean diameter of the papular reactions being evaluated

The intradermal and epicutaneous challenge exposure with the test chemical were negative. Hence, the test chemical was considered to be not sensitizing to the skin of guinea pigs in the Draize test.

The results of the Draize test are supported by a Guinea Pig Maximization Test (GPMT) to assess the dermal sensitization potential of the test chemical

On day 0, the animals were injected intradermally with 0.1ml of a 5% solution of the compound, with 0.1ml of a 5% emulsion of the compound in FCA and with 0.1 ml of FCA alone, each injection was given twice. In addition, on day 8 the compound, dissolved in petrolatum upto 25% was applied to a clipped skin area of the neck and kept under occlusive bandage for 2 days (total dose 20mg intradermally plus 250mg epicutaneously). On day 21, an occlusive patch test with the compound in petrolatum was applied to the flank for 24 hours and the reactions were read 24 and 48 hours after removing the patch

The occlusive challenge patch was negative. Epicutaneous tests 2 and 3 weeks later became positive in 1/8 and 3/8 animals respectively. No sensitisation with the test chemical can be detected by the Maximization test.

These results are further supported by an Open Epicutaneous test conducted in guinea pigs (male and female) to determine the skin sensitization potential of the test chemical.

The pretest was performed to determine the primary irritating threshold concentration of test substances at various concentrations (e.g, 100, 30, 10 and 3%). In this test, a single application of 0.025 ml of each test concentration was simultaneously performed on one of the areas measuring 2 cm2 of the flank skin previously clipped and marked with a circular stamp. Reactions are read 24 h after the application of the test material.  

On day 1 during induction, 0.1 ml of undiluted test chemical was applied to an area measuring 8 cm2 on the clipped flank skin of the guinea pigs. The applications are repeated daily for 3 weeks or done 5 times weekly during 4 weeks, usually on the same skin sites. The application sites were left uncovered and the reactions, if continuous daily applications were performed, can be read 24 h after each application, or at the end of each week. To determine whether or not contact sensitization was induced, all groups of guinea pigs previously treated for 21 days, as well as 10 untreated, or only pretreated with the vehicle, controls are tested on days 21 and 35 on the contralateral flank with the test material. This test was performed by applying with a pipette 0.025 ml of chemical to skin areas measuring 2 cm2. The reactions were read after 24, 48 and/or 72h.

0.025ml of minimal irritating concentration failed to cause any dermal reactions in the challenge exposure. Hence, the test chemical can be considered to be not sensitizing to skin.

The above studies are further supported by a skin sensitization study of the test chemical was carried out in 10 Inbred Hartley strain albino the guinea pigs of to determine its sensitization potential according modified Draize sensitization test. The preliminary irritation tests were done in guinea pigs to determine concentrations suitable for sensitization testing [injection challenge concentration(ICC) and application challenge concentration(ACC) ]

In the induction phase,the total dose was administered on one occasion as 4 intradermal injections, each 2.5 times the ICC (2.5X 0.25). Fourteen days later each animal was challenged intradermally in one flank and topically in the other with 0.1 ml aliquots of test substance at the respective ICC and ACC (0.25 and 10 respectively). Twenty-four hours later the reactions were observed. In the absence of sensitization reactions at first challenge the induction and challenge procedures were repeated, andapparent sensitization reactions confirmed 7 days later by a second challenge with controls included. Based upon the second challenge, it can be concluded that the test chemical was considered to be non-sensitizing to the skin of albino Hartley guinea pigs at 0.25% ICC and 10% ACC concentrations.

The above guinea pig studies are supported by a Human Maximization test performed to evaluate the dermal sensitization potential of the test chemical.

5% test chemical (3450microgram/cm2) in petrolatum was applied under occlusion to the same site on the forearms or backs of 25 subjects for five alternate-day 48-h periods. Patch sites were pre-treated for 24 h with 5% aqueous sodium lauryl sulfate (SLS) under occlusion. Following a 10–14 day rest period, challenge patches were applied under occlusion to fresh sites for 48 h. Challenge applications were preceded by a 60 min SLS treatment (10%). Reactions were read at patch removal and again at 48 and 72 h.

In a human Maximization study involving 25 subjects, there were no positive reactions to 5% test chemical (3450microgram/cm2) in petrolatum.

Hence, the test chemical can be considered to be not sensitizing to skin.

Eventhough the results of the various studies on guinea pigs and humans claim that the test chemical was not sensitizing to skin, but the results of the LLNA and other studies on guinea pigs claim otherwise. Taking into consideration, all these parameters, the test chemical can be considered to be sensitizing to skin. Comparing the above annotations with the criteria of CLP Regulation, the test chemical can be classified under the category “Skin Sensitizer 1”.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Eventhough the results of the various studies on guinea pigs and humans claim that the test chemical was not sensitizing to skin, but the results of the LLNA and other studies on guinea pigs claim otherwise. Taking into consideration, all these parameters, the test chemical can be considered to be sensitizing to skin. Comparing the above annotations with the criteria of CLP Regulation, the test chemical can be classified under the category “Skin Sensitizer 1”.