1-phosphonobutane-1,2,3,4-tetracarboxylate and 2-phosphonosuccinate sodium salts

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2014-12-15 to 2015-02-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-compliant study performed according to an internationally-recognized guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

yes

Remarks:

Max T° at 23.5°C instead of 23°C. Laboratory bedding Grade 5

GLP compliance:

yes (incl. QA statement)

Remarks:

2012-03-12

Type of study:

guinea pig maximisation test

Test material

Test material information

Constituent 1

Test material form:

other: liquid

Details on test material:

Name: DP1268

In vivo test system

Test animals

Species:

guinea pig

Strain:

other: (LAL/HA/BR)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: LAB-ÁLL Bt. Budapest, 1174 Hunyadi u. 7.
- Age at study initiation: 8-9 weeks at onset the treatment
- Weight at study initiation: 464 – 503 g
- Housing: Animals were housed in macrolon cages size IV, with 5 animals/cage to allow socialization
- Diet (e.g. ad libitum): Animals received Cunigra Diet for Rabbits, produced by Bonafarm-Bábolna Takarmány Ltd., Hungary during the main test, ad libitum. This diet is classified as being suitable for guinea pigs as the vitamin D level is high enough to meet the needs of this species. This is the diet used by the breeder/supplier and animals are fully adapted to this diet on arrival.
- Water (e.g. ad libitum): Animals received tap water from municipal supply as for human consumption, containing 50 mg/100 mL ascorbic acid, ad libitum. The drinking water is routinely analysed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
- Acclimation period: 34 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6 – 23.5 °C
- Humidity (%): < 24 – 41 %
- Air changes (per hr): 15-20 air exchange/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily from 6 a.m. to 6 p.m. (artificial light)

IN-LIFE DATES: From 2015-01-06 to 2015-01-30

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

physiological saline

Induction 2

Concentration / amount:

Intra-dermal Induction Exposure (Main study I): 5% (w/v in Saline
Dermal Indicution Exposure (Main Study II): 100% undiluted
Challenge Epxosure (Main study III): 100% undiluted and 50% w/v in saline (Safeguared dose)

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

physiological saline

Challenge 2

Concentration / amount:

Intra-dermal Induction Exposure (Main study I): 5% (w/v in Saline
Dermal Indicution Exposure (Main Study II): 100% undiluted
Challenge Epxosure (Main study III): 100% undiluted and 50% w/v in saline (Safeguared dose)

No. of animals per dose:

Control: 5 animals
Treated group: 10 animals

Details on study design:

RANGE FINDING TESTS:
The concentration of test item to be used for each induction exposure should be well tolerated systemically and should be the highest to cause no more than mild-tomoderate skin irritation. The concentration used for the challenge exposure should be the highest non-irritant dose.

The day prior to the test, the hair was removed from the right and left surface of the animals (approximately 5 x 5 cm). The hair removal was performed carefully to ensure animals are closely shaven.

A series of test item concentrations was tested to identify the primary irritation following intra-dermal injection and dermal application: 0.5, 1, 2.5 and 5% (w/v)
concentrations were used for intra-dermal injection and 25, 50, 75% (w/v) and 100% (undiluted) for dermal application. Local effects were examined and scored 1, 24, 48 and 72 hours after treatment and/or patch removal. Skin effects were scored for erythema and oedema, any other observations of changes to the skin was recorded.

For the intra-dermal application, 0.1 mL per concentration was injected intra-dermally into the hair free skin of the flanks. One concentration was injected on the right side and another concentration on the left side of the animals. Each concentration was injected in duplicate. Two animals were used per concentration.
It was found that concentrations of 0.5, 1, 2.5 and 5% (w/v) in saline produced no reaction (scores 0-0) in the skin of guinea pigs at the 1, 24, 48 and 72 hours
observation.

For the dermal application, approximately 0.5 mL per concentration was applied onto the clipped and shaved skin of the animals. A closed patch exposure was performed by means of an occlusive bandage using similar treatment procedures as for the main study. One concentration was used on the right side and another concentration on left side of the animals. Two animals were used per concentration. Time of exposure was 48 hours.

It was found that 0.5 ml of the test item formulations at concentrations of 100%v(undiluted), 75, 50 and 25% (w/v) produced no reaction (scores 0-0) on the skin of guinea pigs.

On the basis of results of the Preliminary Dose Range Finding Study, the 5% (w/v) concentration was used for intra-dermal treatment and 100% (undiluted) was used for dermal induction treatment.

Control animals were treated with saline.

For the challenge exposure, the 100% (undiluted) concentration was used as challenge dose and a concentration of 50% (w/v) as safeguard dose.

MAIN STUDY
A. INDUCTION EXPOSURE (2 phases)

Main Study I: Intra-dermal Induction Exposure
The day before the treatment, an area of 5 x 5 cm2 on the scapular region of animals was clipped free of hair and shaved.

Test group:
A series of three injections was administered on each side of the scapular region of treatment group animals, as follows, resulting in six injections per animal:
o 2 injections with 0.1 mL of Freund's Complete Adjuvant mixed with saline (1:1) (v/v),
o 2 injections with 0.10 mL of the test item in saline at 5% (w/v) concentration,
o 2 injections with 0.1 mL of test item in 5% (w/v), formulated in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and saline.
Control group:
The control animals were treated similarly as the test group; however, the vehicle without the test item was used for injections as follows:
o 2 injections with 0.1 mL mix of Freund's Complete Adjuvant and saline (1:1) (v/v),
o 2 injections with 0.1 mL of saline,
o 2 injections with 0.1 mL of 50% formulation of saline in a 1:1 mixture (v/v) of Freund's Complete Adjuvant and saline.

Main study II: Dermal Induction Exposure
The same inter-scapular region which received the intradermal injections, were used for dermal induction exposure.
Since the test item was not skin irritant in the Preliminary Dose Range Finding Study, the test area was painted with 0.5 mL of 10 % sodium dodecyl sulphate in Vaseline 24 h prior to the topical induction application, in order to create a local irritation.

Seven days after the intra-dermal injections, the same hair-free scapular area (approximately 6x8 cm) was treated. A 2.5x2.5 cm sterile gauze patch (4 layers of porous gauze pads) was saturated with approximately 0.5 mL of the test item at 100% (undiluted) concentration (the highest dose found non-irritant in the preliminary dose range finding study) and placed over the injection sites.
The control group was treated with 0.5 mL saline.

B. CHALLENGE EXPOSURE
Main study III: Challenge Exposure
Two weeks after the topical induction application, the animals were exposed to a dermal challenge dose. Approximately twenty four hours before the treatment, the hair was removed from an area of approximately 6x8 cm on the left and right flank of each animal. A 5x5 cm patch of sterile gauze patch was saturated with the test item at 100% (undiluted) concentration and applied to the left flank of all animals (both the test and the control). The right shaved flank area of all animals was treated with a 50% dilution of the maximum dermal challenge dose.

The volume of formulated test item was approximately 0.5 mL. Treatment was as indicated in point 3.5.2.2 (Closed Patch Test). The time of the exposure was 24 hours. After the patch removal any remaining test item was removed with a wet gauze swab

Positive control substance(s):

yes

Remarks:

2-Mercaptobenzothiazole

Results and discussion

Positive control results:

RELIABILITY STUDY
The sensitivity and reliability of the experimental procedure is assessed twice a year by use of items which are known to have moderate skin sensitisation properties such as 2-Mercaptobenzothiazole (OECD 406, adopted 17 July 1992 chapter 10., 11.).
The results of the latest reliability check (Study Code: 14/416-104T) are summarised here below.
 
START OF EXPERIMENT :                                   07 October 2014
END OF EXPERIMENT :                                       31 October 2014
FINAL REPORT :                                                 17 December 2014
 
The selection of dose levels was made on the basis of the previous reliability study.
Intra-dermal induction exposure: 1% (w/v)
Dermal induction exposure: 75% (w/v)
Challenge treatment: 50% (w/v)
 
SUMMARY OF THE RELIABILITY STUDY
Challenge with reference item 2-Mercaptobenzothiazole resulted in a positive response in test animals previously sensitised. The net response values at the 24 and 48 hours observations represented an incidence rate of 90% and 80% and net score values of 0.90 and 0.80 respectively. In the control animals no visible changes were found either at the 24 or 48 hours examinations following challenge with the
reference item.
 
The dermal scores represented discrete erythema (score 1) developed on the skin of sensitised guinea pigs.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100%

No. with + reactions:

0

Total no. in group:

0

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 0.0.

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100%

No. with + reactions:

0

Total no. in group:

0

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 0.0.

Any other information on results incl. tables

OBSERVATIONS DURING INDUCTION and CHALLENGE PHASE

 

	N° animal	Induction phase										Challenge phase
		Day 2		Day 10		Day 11		Day 12		Day 13		Day 24		Day 25
		E	O	E	O	E	O	E	O	E	O	E	O	E	O
Control	117	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	128	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	131	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	133	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	137	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Treated	118	0	0	1	0	0	0	0	0	0	0	0	0	0	0
	120	0	0	1	0	0	0	0	0	0	0	0	0	0	0
	126	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	127	0	0	1	0	0	0	0	0	0	0	0	0	0	0
	134	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	135	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	136	0	0	1	0	0	0	0	0	0	0	0	0	0	0
	138	0	0	1	0	0	0	0	0	0	0	0	0	0	0
	139	0	0	1	0	0	0	0	0	0	0	0	0	0	0
	140	0	0	1	0	0	0	0	0	0	0	0	0	0	0

O: Oedema

E: Erythema

0: No erythema or Oedema

1: Very slight erythema (Barely perceptible)

BODY WEIGHT

 

	N° Animal	Day -1	Day 7	Day 14	Day 21	Day 25
Control	117	501	523	541	555	584
	128	483	501	515	531	556
	131	495	498	556	565	591
	133	481	520	559	552	574
	137	464	467	502	501	510
Mean		484.4	501.8	534.6	540.8	563.0
Standard Deviation		14.3	22.4	25.2	25.5	32.4
Treated	118	484	499	530	557	541
	120	503	493	554	576	579
	126	503	517	557	598	614
	127	467	512	551	598	583
	134	465	487	530	557	561
	135	489	512	517	551	547
	136	479	526	516	550	542
	138	474	515	546	580	592
	139	492	530	536	583	567
	140	487	517	546	574	580
Mean		484.3	510.8	538.3	572.4	570.6
Standard Deviation		13.3	13.8	14.8	18.0	23.6

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Migrated information

Conclusions:

Challenge with test item DP1268 evoked no positive responses in the test animals previously sensitised with the test item or in the control group. The net response value represented an incidence rate of 0 % and the net score value of 0.00.

In conclusion, under the conditions of the present assay the test item DP1268 was shown to have no sensitisation potential and classified as a non-sensitizer

Executive summary:

The sensitising potential of DP 1268 was investigated in a guinea pig maximization test comparable to standard conditions of the OECD Guideline 406.

Intradermal induction was performed with a test concentration of 5% followed by a 48-hour occlusive patch with 100% DP 1268 seven days later. After a 14-day interval, challenge was performed by application of an occlusive chamber with 100% DP 1268 to a prepared flank for 24 hrs.

No sensitzing activity of DP 1268 was indicated in the guinea pig maximization test.