Alkyl phosphites

Skin sensitisation

Administrative data

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:

other: GLP study conducted in accordance to EU/OECD guidelines. Some details on test material are missing.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

and OECD 406

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

This study was conducted in 1996, pre-dating the OECD 429 Guideline and REACH Legislation, therefore no justifcation is required for the use and inclusion of this non-LLNA method.

Test material

Test material information

Constituent 1

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
- Age at study initiation: Approximately 7 weeks
- Weight at study initiation: 320 to 454 grams
- Housing: Single housed in suspended steel wire mesh with aborbent paper below cages.
- Diet: PMI certified Guinea Pig Diet 5026 (ad libitum).
- Water: ad libitum
- Acclimation period: 21 days (animals were examined for viability at least once daily).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.3 to 21.6
- Humidity (%): 40 to 70%
- Air changes (per hr): Not stated
- Photoperiod (hrs dark / hrs light): 12 hours light (07:00 to 19:00) and 12 hours dark (19:00 to 07:00)

IN-LIFE DATES: From: To: 1996-05-28 to 1996-06-24

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

peanut oil

Induction 2

Concentration / amount:

Intradermal Induction (Day 0): 0.1% test material in peanut oil (w/v mixture).

Epicutaneous Induction (Day 7): 5% test material in peanut oil (w/v mixture).

Epicutaneous Challenge (Day 21): 0.05% test material in peanut oil (w/v mixture).

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

peanut oil

Challenge 2

Concentration / amount:

Intradermal Induction (Day 0): 0.1% test material in peanut oil (w/v mixture).

Epicutaneous Induction (Day 7): 5% test material in peanut oil (w/v mixture).

Epicutaneous Challenge (Day 21): 0.05% test material in peanut oil (w/v mixture).

No. of animals per dose:

20

Details on study design:

RANGE FINDING TESTS: A primary irritation test was performed to determine the concentration of the test material to be used for intradermal induction. Three concentrations were tested (0.1, 1.0 and 5.0% w/v). A pair of 0.1 ml injections of the follwoing solutions were intradermally administered to each of 3 sites in the mid dorsal region: Site 1 - diluted FCA, Site 2 - test material in carrier, Site 3 - test material in diluted FCA (two animals per concentration). The injection sites were evaluated 24 and 48 hours post injection.

A primary irritation test was also conducted to determine the concentrations for topical induction and challenge. After pretreatment with two pairs of injections with 50% FCA in reverse osmosis water, four concentrations of the test material (0.1, 1.0, 5.0 and 10% w/w in peanut oil) were topically applied to the flanks of five animals per concentration.

MAIN STUDY

Number and Sex

40 females (nulliparous and non pregnant): 20 females for the treated group and 20 females for the irritation control group.

An additional group of 15 females was used for testing of the positve control material.


Intradermal Induction (Day 0)

Preparation of Animals: An area near the scapula in the mid dorsal region of all animals was clipped on the day prior to intradermal injection of the test material and/or carrier. All animals were clipped using Oster A-5 small animal clipper equipped with size 40 blades.

Administration of Test Material: A pair of 0.1 ml injections of the following mixtures were administered intradermally to each of three sites in the mid dorsal region of all animals near the scapula.

Site 1 - diluted FCA to treated and control groups.

Site 2 - test material in peanut oil to treated group, undiluted peanut oil to the control group.

Site 3 - test material in diluted FCA to the treated group (the concentration was adjusted so that the absolute amount of material was the same as that administered at site 2), peanut oil diluted in FCA to the control group.

The solutions were injected intradermally with an appropriate size syringe and 25 gauge needle. A row of three injections, 0.1 ml of each solution was given along both sides of the dorsal midline for a total of six injections. Solutions for Site 1 and site 2 were injected close together and near the first thoracic vertebra: the solution for Site 3 was injected caudally. All injections were made 1-2 cm from the midline and within the boundaries of the clipped region.

Induction by Occlusive Topical Application (Day 7)

Preparation of Animals

The fur in the previously treated scapular area was reclipped on the day prior to topical application of the test material or carrier.

Administration of Test Material: Seven days after intradermal induction, 0.5 ml of 5% test material diluted in carrier (peanut oil) was applied topically over the previously treated and clipped scapula area of the treated group animals. The irritation control group animals recieved 0.5 ml of carrier alone. The test material and/or carrier was applied to the skin under a 2x4 cm filter paper, covered by a overlapping plastic tape and firmly secured to the torso of the animals with elastic adhesive bandaging. The pads and sleeving were removed from both treated and control group animals after approximately 48 hours. The skin was gently wiped free of remaining test material and/or carrier with dry surgical gauze.

Challenge by Occlusive Topical Application.

Preparation of Animals

The fur in an area on the left and right flank in the abdominal region was clipped on the day prior to challenge dosing of the test material.

Administration of Test Material: Twenty one days after intradermal induction, 0.4 ml of 0.05% test material diluted in carrier (peanut oil) was apllied topically to the clipped area on the left flank and 0.5 ml of carrier alone was applied to the right flank of all treated and ten irritation control group animals. The test material and the carrier each were applied to the respective Hilltop Chamber (Hilltop Research Inc, Cincinnati Ohio) and firmly secured to the torso of the animals with elastic adhesive bandaging. The chambers and sleeves were removed from both treated and control animals after approximately 24 hours and the dose site wiped clean with paper towels and peanut oil to remove any residual material.

Approxiamtely 21 hours after removal of the challenge patch, the dose site was clipped

Challenge controls:

A challenge control group was run in parallel with the treated group.

Positive control substance(s):

yes

Remarks:

2-mercaptobenzothiazole (MBT)

Results and discussion

Positive control results:

Intradermal administration of MBT to the positive control group produced moderate to extreme redness around the injection site and red, tan or brown centres of the injection sites. Topical induction of MBT elicited Grade 0 to ± responses in 7 of 15 animals and grade 1 response in 8 animals at 24 hour observation. At the 48 hour observation 14 of 15 animals exhibited scores of 0 to ± while one animal had a score of 1.

Following challenge increased reactivity to MBT was observed in the majority of animals with dermal responses as high as grade 2 and 3. Supplemental dermal observations for the positive control animals following challenge included desquamation and/or edema. Together these results indicate that the test system responded in an appropriate manner to the positive control.

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

0.05%

No. with + reactions:

3

Total no. in group:

20

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 0.05%. No with. + reactions: 3.0. Total no. in groups: 20.0.

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

0.05%

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 0.05%. No with. + reactions: 0.0. Total no. in groups: 20.0.

Results 3

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0.05%

No. with + reactions:

2

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 0.05%. No with. + reactions: 2.0. Total no. in groups: 10.0.

Results 4

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0.05%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0.05%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 5

Key result

Reading:

1st reading

Hours after challenge:

1

Group:

positive control

Dose level:

3%

No. with + reactions:

15

Total no. in group:

15

Remarks on result:

positive indication of skin sensitisation

Results 6

Key result

Reading:

2nd reading

Hours after challenge:

24

Group:

positive control

Dose level:

3%

No. with + reactions:

15

Total no. in group:

15

Remarks on result:

positive indication of skin sensitisation

Any other information on results incl. tables

RANGE FINDING TESTS

Primary Irritation Test

A primary irritation test was performed to determine the concentration of the test material to be used for intradermal induction. Three concentrations were tested (0.1, 1.0 and 5.0% w/v). A pair of 0.1 ml injections of the follwoing solutions were intradermally administered to each of 3 sites in the mid dorsal region: Site 1 - diluted FCA, Site 2 - test material in carrier, Site 3 - test material in diluted FCA (two animals per concentration). The injection sites were evaluated 24 and 48 hours post injection. All injection sites for the 5% animals developed necrosis in 48 hours. The Site 2 injection site for 1% animals also developed necrosis. Therefore the concentration chosen fo intradermal induction was 0.1% of the test material.

A primary irritation test was also conducted to determine the concentrations for topical induction and challenge. After pretreatment with two pairs of injections with 50% FCA in reverse osmosis water, four concentrations of the test material (0.1, 1.0, 5.0 and 10% w/w in peanut oil) were topically applied to the flanks of five animals per concentration. The animals treated with 10% test material responded with irritation too severe to be used for topical induction. However the animals treated with 5% test material responded with irritation that was too irritating for use in the challenge phase but was sufficiently irritation for topical induction. To determine the challenge concentration, four concentrations of the trst material (0.05, 0.1, 0.5 and 1% w/w) were topically applied to five animals per concentration. Again all animals responded with irritation but the irritation in the 0.05 and 0.1% concentration animals was resolved by the 48 hour evaluation. Since the 0.1% concentration elicited a grade 2 at 24 hours the 0.05% concentration was chosen for the challenge.

Intradermal induction

 

Intradermal administration of 0.1% test material to the scapula area produced slight to moderate redness around the injection site in all 20 treated group animals. However there were no signs of necrosis or eschar, indicating that the injections were well tolerated locally.

The intradermal administration of peanut oil to the irritation control group animals produced results similar to the treated group animals.

 

Topical Induction

Topical administration of 5% test material to the scapula area produced Grade ± to 1 response in 18 of the 20 treated group animals, and Grade 2 or 3 responses in the remaining two animals at the 1 hour evaluation. One animal was observed with blanching. The responses increased slightly by the 24 hour evaluation at which time 13 of 17 animals had responses=1. Necrosis, fissuring and ulceration also were observed at 24 hours.

Topical application of peanut oil to the scapular area produced Grade 0 to ± responses in 19 of 20 control animals and grade 1 erythema in one control group animal at the 1 hour evaluation. The erythema decreased considerably by the 24 hour evaluation.

 

Challenge  

Topical application of 0.05% test material to the treated group animals produced grade 0 to ± responses in 17 of 20 animals and grade 1 response in the remaining three animals at the 24 hour observation. At the 48 hour observation 14 of 20 animals had grade 0 responses while the remaining 6 animals had Grade ± responses.

 

In the irritation control group topical application of 0.05% test material produced grade 0 to ± responses in 8 of 10 animals and grade 1 response in the remaining 2 animals at the 24 hour observation. At the 48 hour observation all responses in the irritation control animals were 0 or ±.

 

Topical application to the treated group animals produced Grade 0 to ± responses in 18 of 20 animals, and grade 1 response in the remaining 2 animals at the 24 hour observation. At the 48 hour observation two animals had grade ± responses while all other scores were 0.

 

In the irritation control group, topical application of peanut oil produced Grade 0 to ± responses in 7 of 10 animals and grade 1 response in 3 animals at the 24 hour observation. At the 48 hour observation all responses were 0 or ±.

Weights and In-life Observations

All animals survived to the schedule termination and showed overall increase in body weight during the test period.

 

Most animals were free of abnormalities throughout the study period. Two treated animals and one irritation control animals were observed with anogenital staining on day 14. However all animals showed no observable abnormalities throughout the rest of the study.

 

Positive Control

 

Intradermal administration of MBT to the positive control group produced moderate to extreme redness around the injection site and red, tan or brown centres of the injection sites. Topical induction of MBT elicited Grade 0 to ± responses in 7 of 15 animals and grade 1 response in 8 animals at 24 hour observation. At the 48 hour observation 14 of 15 animals exhibited scores of 0 to ± while one animal had a score of 1.

Following challenge increased reactivity to MBT was observed in the majority of animals with dermal responses as high as grade 2 and 3. Supplemental dermal observations for the positive control animals following challenge included desquamation and/or edema. Together these results indicate that the test system responded in an appropriate manner to the positive control.

 

Analytical Chemistry

Test substance in peanut oil was shown to the uniform at 0.05% and 5% w/v. Stability data indicated that the test substance in peanut oil was stable for two days at room temperature. Concentration verification analysis indicated that test substance in peanut oil was within 9% of the nominal concentration.

Satisfactory uniformity was observed for 2-MBT in peanut oil. Concentration verification analysis indicated that 2-MBT in peanut oil was within 9% of the nominal concentration.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

All animals survived to termination and displayed increases in body weight over their Day 0 values. Clinical signs were limited to anogenital staining in 3 animals (2 in the treated group and 1 in the control group) on Day 14. These signs were considered related to the topical induction procedure and not the test substance.

Intradermal induction with 0.1% test substance did not produce necrosis or eschar. Topical induction with 5.0% test substance produced slight patchy to severe irritation. Blanching fissuring, ulceration and necrosis were also observed following topical induction.

Topical challenge with 0.05% test substance elicited dermal scores ranging from 0 to 1 in both the treated and irritation control animals at 24 hours. The vehicle peanut oil produced a similar level of dermal response at 24 hours. At 48 hours all score in all animals were 0 or ±.

The positive control produced increased responses in the majority of guinea pigs at challenge, indicating that the test system responded in an appropriate manner.

In conclusion based on challenge scores the test substance did not display evidence of contact sensitization in the guinea pig.

Executive summary:

The allergic contact sensitization of the test substance was evaluated using the Guinea Pig Maximization Test as set out by EU guideline B.6 and OECD guideline 406. Twenty female Hartley Albino guinea pigs received intradermal induction, topical induction and topical challenge with the test substance.

Primary rangefinding test were conducted to determine appropriate concentrations of the test substance to be used in the three phases of dosing. Based on the results of these tests, concentrations of 0.1%, 5.0% and 0.05% were chosen for intradermal induction, topical induction and topical challenge respectively.

A positive control group consisting of 15 guinea pigs was included in the study. These animals received 2-mercaptobenzothiazole (MBT) in peanut oil at concentrations of 3.0%, 25% and 0.5% for intradermal induction, topical induction and topical challenge respectively.

Dermal responses were evaluated 1 and 24 hours after the intradermal induction using descriptive terminology. Dermal responses were evaluated approximately 1 and 24 hours (Days 9 and 10 respectively) after removal of the topical induction patch and 24 and 48 hours (days 23 and 24 respectively) after removal of the topical challenge patch. Observations were made of the nature, onset, severity and duration of clinical signs after dosing on day 0 and on day 7, 14, 21 and 27. After final observations and weighing on day 27 all animals were sacrificed by carbon dioxide asphyxiation and discarded without further evaluation.

All animals survived until termination and displayed an increase in body weight over their day 0 values. Clinical signs were limited to anogenital staining in three animals (2 in the treated group and 1 in the irritation group) on day 14. These signs were considered related to the topical induction procedure and not the test substance.

Intradermal induction with 0.1% test substance did not produce necrosis or eschar. Topical induction with 5% test substance produced slight patchy to severe irritation. Blanching, fissuring, ulceration and necrosis were also observed following topical induction.

Topical challenge with 0.05% test substance elicited dermal scores ranging from 0 to 1 in both the treated and the irritation control animals at 24 hours. The vehicle peanut produced a similar level of dermal response at 24 hours. At 48 hours all scores in all animals were 0 or ±.

The positive control produced increased responses in the majority of guinea pigs at challenge indicating the test system responded in an appropriate manner.

In conclusion based on challenge scores, the test substance did not display evidence of contact sensitization in the guinea pig.