Benzoic acid, 2-hydroxy-, mono-C14-18-alkyl derivs., calcium salts (2:1)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002-08-21 to 2002-10-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

Buehler test

Justification for non-LLNA method:

It is considered that this type of substance shows surfactant properties and so would be likely to demonstrate false positives or potency in an LLNA assay. Therefore a more appropriate Buehler assay had been conducted some 10 years earlier to a suitable guideline and this test was considered valid and reliable.

Test material

Test material information

Constituent 1

Reference substance name:

Benzoic acid, 2-hydroxy-, mono-C14-18-alkyl derivs., calcium salts (2:1)

EC Number:

931-276-9

Cas Number:

114959-46-5

Molecular formula:

See information in Section 1.2.

IUPAC Name:

Benzoic acid, 2-hydroxy-, mono-C14-18-alkyl derivs., calcium salts (2:1)

Details on test material:

- Physical state: Black liquid
- Lot/batch No.: Sample #80002/01 (MRD-02-463)
- Expiration date of the lot/batch: August 2007
- Storage condition of test material: Room temperature

In vivo test system

Test animals

Species:

guinea pig

Strain:

Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Kingston Facility, Stone Ridge, NY
- Age at study initiation: 5-6 weeks
- Weight at study initiation: 318-394 g
- Housing: Singly in suspended stainless steel and wire mesh cages with absorbant paper below
- Diet (e.g. ad libitum): Guinea Pig Diet 5026 (PMI Nutrition International LLC, Richmond, Indiana) ad libitum. Availability of feed was checked at least once daily for all animals.
- Water (e.g. ad libitum): Site deionised water ad libitum via automated system. Availability of water was checked at least once daily for all animals.
- Acclimation period: 15 days during which animals were examined for viability at least once per day

ENVIRONMENTAL CONDITIONS
- Temperature: 64-72 degrees Fahrenheit monitored continuously
- Humidity: 30-70 % relative humidity monitored continuously
- Air changes (per hr): no data
- Photoperiod: Approximately 12 hours light (06:00 to 18:00) and 12 hours dark (18:00 to 06:00) by automatic timer

IN-LIFE DATES: From: 2002-08-28 To: 2002-10-04

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

epicutaneous, occlusive

Vehicle:

other: The test substance was dosed as received for the topical induction and mixed with olive oil to form a 20 % mixture for the challange and rechallenge applications

Induction 2

Concentration / amount:

Neat for the topical challenge and 20 % in olive oil for challenge and rechallenge procedures

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

other: The test substance was dosed as received for the topical induction and mixed with olive oil to form a 20 % mixture for the challange and rechallenge applications

Challenge 2

Concentration / amount:

Neat for the topical challenge and 20 % in olive oil for challenge and rechallenge procedures

No. of animals per dose:

20 females (nulliparous and non-pregnant)

Details on study design:

INDUCTION BY OCCLUSIVE TOPICAL APPLICATION (Days 0, 7 and 14)

Animals were identified by monel ear tags and corresponding cage identification.

The scapular region of the treated animals was clipped on the day prior to each topical induction of the test substance. Treated animals were clipped using Oster small animal clippers equipped with size 40 blades. The irritation control group was not clipped.

Induction doses were administered on Days 0, 7 and 14. The neat test substance (0.4 mL) was applied topically to the previously clipped left scapular area of the treatment group animlas. The test substance was applied on a 1.5 x 1.5 inch First Aid Sheer Adhesive Bandgage (ACME/Charlston) and firmly secured to the torso of the animals with elastic adhesive bandaging (Elastopast or Elastikon). The use of the adhesive bandaging ensured the contact of the test substance with skin and slightly restricted animal movement.

The bandaging was removed from the treated animals after approximately 6 hours. The residual test substance was removed gently at the time of bandage removal with paper towels moistened with peanut oil.

The irritation control group animals remained untreated.

CHALLENGE BY OCCLUSIVE TOPICAL APPLICATION (Day 28)

The fur in a naive area on the right flank region was clipped for all treated animals and the irritation control animals on the day prior to challenge dosing.

Twenty eight days after the initial topical induction, 0.4 mL of the 20 % test substance in olive oil was applied topically using a Hilltop Chamber (Hilltop Research Inc, Cincinnati, Ohio) to the clipped area of the right flank of all treated and irritation control group animals. The test substance was applied to the chamber and firmly secured to the torso of the animals with elastic adhesive bandaging (Elastoplast or Elastokon). The use of adhesive bandaging ensured contact of the test substance with the skin and slightly restricted the movement of the animals.

The Hilltop Chamber and sleeves were removed from both treated and control animals after approximately 6 hours. The challenge area was gently wiped free of remaining test substance with paper towels moistened with olive oil, taking care not to alter the existing response or the integrity of the epidermis.

On Day 29, approximately 21 hours after removal of the challenge patches, the challenge areas were gently clipped to facilitate grading of dermal irritation. Dermal responses were evaluated approximately 24 and 48 hours after removal of the challenge patches (Days 29 and 30 respectively) according to the method of scoring presented in Key A (attached).

RECHALLENGE BY OCCLUSIVE TOPICAL APPLICATION (Day 35)

A naive area on the left flank region was clipped for all treated animals on the day prior to rechallenge dosing.

Thirty five days after initial topical induction, 0.4 mL of 20 % test substance in olive oil was topically applied to the clipped area on the left flank of all treated and irritation control group animals. The test substance was applied on a Hilltop Chamber (Hilltop Research Inc, Cincinnati, Ohio) and firmly secured to the torso of the animals with elastic adhesive bandaging (Elastoplast or Elastikon). The Hilltop Chamber and sleeving were removed from both treated and control animals after approximately 6 hours and the rechallenge area was gently wiped free of remaining test substance and/or carrier with paper towels moistened with olive oil without altering existing response or the integrity of the epidermis.

On Day 36, approximately 21 hours after removal of the rechallenge patch, the rechallenge area was gently clipped. Dermal responses were evaluated at approximately 24 and 48 hours after removal of the rechallenge patch (Days 36 and 37 respectively) according to the method of scoring presented in Key A (attached).

EXPERIMENTAL EVALUATION
The animals were examined for viability twice daily on Monday to Friday, and once daily on weekends and/or holidays.

Clinical observations were made as to the nature, onset, severity and duration of toxicological signs after dosing on Day 0 and on Days 7, 14, 21, 28 and 35, as well as prior to sacrifice on Day 37. Cage-side observations were performed on the days that clinical observations were not performed.

Bodyweights were recorded pretest for sorting purposes, prior to initiation on Day 0, and on Days 7, 14, 21, 28, 35, and prior to sacrifice on Day 37.

Dermal responses were evaluated approximately 24 and 48 hours after initiation of each of the induction doses and approximately 24 and 48 hours after removal of the challenge patches (Days 36 and 37 respectively). All scoring was performed according to the method presented in Key A (attached).

On Day 37, all remaiing animals were observed, weighed, sacrificed by carbon dioxide asphyxiation and discarded without further examination.

DATA INTERPRETATION
The incidence of sensitisation was determined using the following methods:

a) The incidence of scores in the control and treated groups for each evaluation interval were compared to determine if there was a difference between groups. Greater weight was given to the 48 hour interval.
b) Responses graded +/- were not considered indicative of sensitisation unless the difference in incidence between the control and treated group was overwhelming and the incidence of other scores were minimal in both groups.
c) The 24 and 48 hour responses for each individual treated and control animal were compared. Sensitisation was considered to have occurred if treated animals that exhibited responses graded greater than or equal to 1 at the 24 hour interval continued to be graded greater than or equal to 1 at the 48 hour interval. However, if a similar response was noted in the control group then the response was probably irritation.
d) Edema, eschar, fissuring, or ulceration noted in a treated animal was considered indicative of sensitisation unless noted in the control animals.
e) The incidence of animals showing signs of sensitisation was determined based on the criteria described above. The normalised incidence of control animals (i.e. control incidence times 2) displaying the same sign (if any) was substracted from the incidence in the treated group. The percentage of animals showing signs of sensitisation was calculated and reported.

Challenge controls:

10 females (nulliparous and non-pregnant)

Positive control substance(s):

yes

Remarks:

See Appendix F (attached)

Results and discussion

Positive control results:

The positive control material, MBT, produced evidence of contact sensitisation following challenge. During induction, topical application of MBT elicited dermal irritation of Grade +/- in one animal at the 24 hour evaluation after the first induction application. Following challenge, all 20 positive control animals displayed responses that were considered indicative of contact sensitisation. At the 24 hour challenge evaluation, irritation of Grade +/- was noted in two of the 10 irritation control animals and Grade 1 irritation in one animal. No signs of irritation were noted at the 48 hour evaluation for the irritation control animals. Results are presented in Appendix F (attached)

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

Induction: neat test material; Challenge: 20 % test material in olive oil

No. with + reactions:

7

Total no. in group:

20

Clinical observations:

13 animals grade 0; 7 animals grade +/-

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: Induction: neat test material; Challenge: 20 % test material in olive oil. No with. + reactions: 7.0. Total no. in groups: 20.0. Clinical observations: 13 animals grade 0; 7 animals grade +/-.

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

Induction: neat test material; Challenge: 20 % test material in olive oil

No. with + reactions:

3

Total no. in group:

20

Clinical observations:

17 animals grade 0; 3 animals grade +/-

Remarks on result:

other: see Remark

Remarks:

Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: Induction: neat test material; Challenge: 20 % test material in olive oil. No with. + reactions: 3.0. Total no. in groups: 20.0. Clinical observations: 17 animals grade 0; 3 animals grade +/-.

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

Induction: no treatment; Challenge: 20 % test material in olive oil

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

10 animals grade 0

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: Induction: no treatment; Challenge: 20 % test material in olive oil. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: 10 animals grade 0.

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

Induction: no treatment; Challenge: 20 % test material in olive oil

No. with + reactions:

1

Total no. in group:

10

Clinical observations:

9 animals grade 0; 1 animal grade +/-

Remarks on result:

other: see Remark

Remarks:

Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: Induction: no treatment; Challenge: 20 % test material in olive oil. No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: 9 animals grade 0; 1 animal grade +/-.

Results 5

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

20 % test material in olive oil

No. with + reactions:

9

Total no. in group:

20

Clinical observations:

11 animals grade 0; 6 animals grade +/-; 3 animals grade 1

Remarks on result:

other: see Remark

Remarks:

Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: 20 % test material in olive oil. No with. + reactions: 9.0. Total no. in groups: 20.0. Clinical observations: 11 animals grade 0; 6 animals grade +/-; 3 animals grade 1.

Results 6

Reading:

rechallenge

Hours after challenge:

48

Group:

test chemical

Dose level:

20 % test material in olive oil

No. with + reactions:

9

Total no. in group:

20

Clinical observations:

11 animals grade 0; 8 animals grade +/-; 1 animal grade 1

Remarks on result:

other: see Remark

Remarks:

Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 20 % test material in olive oil. No with. + reactions: 9.0. Total no. in groups: 20.0. Clinical observations: 11 animals grade 0; 8 animals grade +/-; 1 animal grade 1.

Results 7

Reading:

rechallenge

Hours after challenge:

24

Group:

negative control

Dose level:

20 % test material in olive oil

No. with + reactions:

2

Total no. in group:

10

Clinical observations:

8 animals grade 0; 2 animals grade +/-

Remarks on result:

other: see Remark

Remarks:

Reading: rechallenge. . Hours after challenge: 24.0. Group: negative control. Dose level: 20 % test material in olive oil. No with. + reactions: 2.0. Total no. in groups: 10.0. Clinical observations: 8 animals grade 0; 2 animals grade +/-.

Results 8

Reading:

rechallenge

Hours after challenge:

48

Group:

negative control

Dose level:

20 % test material in olive oil

No. with + reactions:

1

Total no. in group:

10

Clinical observations:

9 animals grade 0; 1 animal grade +/-

Remarks on result:

other: see Remark

Remarks:

Reading: rechallenge. . Hours after challenge: 48.0. Group: negative control. Dose level: 20 % test material in olive oil. No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: 9 animals grade 0; 1 animal grade +/-.

Results 9

Reading:

1st reading

Hours after challenge:

48

Group:

positive control

Dose level:

25% MBT in peanut oil

No. with + reactions:

20

Total no. in group:

20

Remarks on result:

positive indication of skin sensitisation

Results 10

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

25%MBT in peanut oil

No. with + reactions:

18

Total no. in group:

20

Any other information on results incl. tables

INDUCTION (see Appendix A, attached)

Topical induction of the neat test substance during the induction phase produced irritation in five of the treated animals. Grade ± irritation was noted in two animals on Day 1 and in one animal at the Day 9 evaluation. Grade 1 irritation was noted in two animals at the Day 1 evaluation.

CHALLENGE (see Table 1, attached)

Challenge with the 20 % test substance in olive oil produced Grade ± responses in six treated animals and Grade 1 responses in seven treated animals at the Day 29 evaluation. The irritation control group was free of irritation at the Day 29 evaluation. The responses noted at the Day 30 evaluation were Grade ± in one irritation control animal and three treated group animals.

RECHALLENGE (see Table 2, attached)

The rechallenge with 20 % test substance in olive oil produced Grade ± responses in six treated animals and Grade 1 responses in three treated animals at the Day 36 evaluation. Grade ± irritation was noted for two animals in the irritation control group on Day 36. The responses noted at the Day 37 evaluation were Grade ± in one irritation control animal, Grade ± in eight treated group animals and Grade 1 in one treated group animal.

BODY WEIGHTS AND IN-LIFE OBSERVATIONS (see Appendices D and E, attached)

All animals showed an overall increase in body weight during the test period, and were free of observable abnormalities throughout the test period.

PRIMARY IRRITATION TEST (see Appendix G, attached)

During the primary irritation test conducted prior to study initiation, the neat (100 %) test substance elicited dermal irritation of Grade 1 in three animals and Grade ± in one animal at the 24 hour evaluation. Grade ± irritation was also noted for one animal each at the 10 and 50 % treatment sites at 24 hours. Grade ± irritation was noted for three animals at the 100 % site at 48 hours.

During the primary irritation test conducted the week prior to the challenge, 40 and 50 % test substance in oilve oil elicited dermal irritation of Grade ± in four animals each at the 24 hour evaluation. The application of 30 % test substance in olive oil elicited irritation of Grade ± in two animals at 24 hours. At the 48 hour evaluation Grade ± irritation was noted for three animals and Grade 1 irritation for one animal at both the 40 and 50 % sites. Grade ± irritation was noted for two animals at the 30 % site at 48 hours. The 20 % site was free of irritation at both the 24 and 48 hour evaluations.

Based on these results, it was decided to administer the neat test substance for induction and 20 % test substance for the challenge.

Experimental Groups

	Treated Group	Irritation Control Group
Number of animals (females)	20	10
Day 0 (0.4 mL)	Neat test substance	Untreated
Day 7 (0.4 mL)	Neat test substance	Untreated
Day 14 (0.4 mL)	Neat test substance	Untreated
Day 28 (0.4 mL)	20 % test substance in olive oil (w/v)	20 % test substance in olive oil (w/v)
Day 35 (0.4 mL)	20 % test substance in olive oil (w/v)	20 % test substance in olive oil (w/v)

Applicant's summary and conclusion

Conclusions:

The test substance produced signs of sensitisation during rechallenge in only two animals and, consequently, 10 % of the animals were considered to have been sensitised to the test substance. This level is insufficient to warrant classification.