Sodium sulphate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04-Aug-2010 to 13-Sep-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no minor deviations from standard test guidelines and/or minor methological deficiencies which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

According to ECHA guidance R.7.a "Guidance on Information Requirements and Chemical Safety Assessment" (December 2016) tests other than LLNA generated or initiated before 11 October 2016 are acceptable, if these tests provide clear results that are adequate for classification. This well documented guideline test has been conducted in 2010 and thus, further in vivo testing is not necessary.

Test material

Test material information

Constituent 1

Reference substance name:

Sodium sulphate

EC Number:

231-820-9

EC Name:

Sodium sulphate

Cas Number:

7757-82-6

Molecular formula:

Na2SO4

IUPAC Name:

disodium sulfate

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Breeder: Harlan Laboratories B.V., Kreuzelweg 53, 5961 NM Horst / the Netherlands
- Number of Animals for Pretest / Main Test: 3 males / 15 males. Animals of either sex are acceptable for use according to guidelines Commission Regulation (EC) No 440/2008, B.6 and OECD 406.
- Age at Pretest Start / Beginning of Acclimatization Period: 4 - 5 weeks
- Body Weight at Pretest Start: Pretest groups: 366 - 389 g
- Body Weight at Beginning of Acclimatization Period: Test and control groups: 347 - 393 g
- Identification: By unique cage number and corresponding individual ear tag.
- Randomization: Selected by hand at time of delivery. No computer generated randomization program.
- Acclimatization: Twelve days for the test and control groups of the main test under standard laboratory conditions after health examination. No acclimatization for the animals of the pretest. Only animals without any visible signs of illness were used for the study. A certificate of health was provided by the animal supplier at animal delivery and included in the raw data.
- Accomodation: Individually in Makrolon type-4 cages with standard softwood bedding (‘Lignocel’ J. Rettenmaier&Söhne GmbH&CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba AG, 4303 Kaiseraugst / Switzerland).
- Diet: Teklad Global Guinea pig diet 2040C, batch no. 24/10 (provided by Provimi Kliba AG, 4303 Kaiseraugst / Switzerland), ad libitum. A haystick 4642, batch no. 54/09 (Provimi Kliba AG, 4303 Kaiseraugst / Switzerland)was also provided for environmental enrichment. Results of analyses for contaminants are archived at Harlan Laboratories Ltd.
- Water: Community tap water from Füllinsdorf, ad libitum. Results of bacteriological, chemical and contaminant analyses are archived at Harlan Laboratories Ltd.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- relative humidity: between 30-70%
- Air changes: 10-15 air changes per hour
- Photoperiod: 12 hours light and 12 hours dark and music played during the daytime light period.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

polyethylene glycol

Remarks:

PEG 300

Induction 2

Concentration / amount:

Intradermal induction: 25% in PEG 300
Epidermal induction: 75% in PEG 300
Epidermal Challenge: 50% in PEG 300

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

polyethylene glycol

Remarks:

PEG 300

Challenge 2

Concentration / amount:

Intradermal induction: 25% in PEG 300
Epidermal induction: 75% in PEG 300
Epidermal Challenge: 50% in PEG 300

No. of animals per dose:

5 control and 10 test for the main test
(1 animal for the intradermal pretest and 2 animals for the epdiermal pretest)

Details on study design:

Induction
1) Intradermal Injection / Performed on Test Day 1
An area of dorsal skin from the scapular region (approximately 6 x 8 cm) was clipped free of hair. Three pairs of intradermal injections (0.1 mL/site) were made just within the boundaries of a 4 x 6 cm area in the clipped region as follows:

Test Group: 1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) The test item at 25% in PEG 300.
3) The test item at 25% in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
Control Group:1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) PEG 300
3) 1:1 (w/w) mixture of PEG 300 in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

2) Epidermal Induction / Performed on Test Day 8
One week after the intradermal injections, the scapular area (approximately 6 x 8 cm) was again shaved prior to epidermal induction. A 2 x 4 cm patch of filter paper was saturated with the test item at 75% in PEG 300 and placed over the injection sites of the test animals. The amount of test item preparation applied was approximately 0.3 g. The patch was covered with aluminum foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The occlusive dressings were left in place for 48 hours. The epidermal application procedure described ensured intensive contact of the test item.

The guinea pigs of the control group were treated as described above with PEG 300 only, applied at a volume of approximately 0.3 mL.

The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema according to the method of Magnusson
and Kligman.

Challenge / performed on Test Day 22
The test and control guinea pigs were challenged two weeks after epidermal induction and treated in the same way.

Two patches (3 x 3 cm) of filter paper were saturated with the test item at the highest tested non-irritating concentration of 50% (applied to the left flank) and the vehicle only (PEG 300 applied to the right flank) using the same method as for the epidermal application. The volume of test item preparation applied was approximately 0.2 mL and a volume of approximately 0.2 mL was used for the vehicle. The dressings were left in place for 24 hours.

TREATMENT METHOD
The animal's fur was shaved with a fine clipper blade just prior to exposure. Intradermal injections or closed patches were applied to the animals as follows:

0.1 mL/site for the intradermal administrations and 0.2 to 0.3 g (or mL) on a patch of filter paper for the epidermal administrations.

The patch was covered by a strip of aluminium foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The occlusive dressing was left in place for 24 (epidermal pretest and epidermal challenge) or 48 hours (epidermal induction).

Identical patching method was used for the epidermal pretest, epidermal induction and epidermal challenge.

Positive control substance(s):

yes

Remarks:

ALPHA-HEXYLCINNAMALDEHYDE

Results and discussion

Positive control results:

The study was performed with 15 (10 test and 5 control) male albino Dunkin Hartley guinea pigs, HsdPoc: DH, SPF, delivered by Harlan Laboratories B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands.

The intradermal induction of sensitization in the test group was performed in the nuchal region with a 3% dilution of the test item in PEG 300 and in an emulsion of Freund's Complete Adjuvant (FCA)/physiological saline. The epidermal induction of sensitization was conducted for 48 hours under occlusion with the test item at 10% in PEG 300 one week after the intradermal induction. The animals of the control group were intradermally induced with PEG 300 and FCA/physiological saline and epidermally induced with PEG 300 under occlusion.

Two weeks after epidermal induction the control and test animals were challenged by epidermal application of the test item at 3% in PEG 300 and PEG 300 alone under occlusive dressing.

Cutaneous reactions were evaluated at 24 and 48 hours after removal of the dressing.

Skin effects in the challenge:
Control Group: No local skin reactions were observed in the control animals when treated with PEG 300 alone. Discrete/patchy erythema was observed in one animal (10%) at the 24-hour reading after treatment with test item at 3% in PEG 300 alone.

Test Group: No local skin reactions were observed in the test animals when treated with PEG 300 alone. Discrete/patchy to moderate and confluent erythema as well as scaling were observed in all ten animals (100%) at the 24- and 48-hour readings after treated with the test item at 3% in
PEG 300. Scaling was observed in nine animals at the 48-hour reading.

No toxic signs were evident in the guinea pigs of the control or test group.

No deaths occurred.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

50% in PEG 300

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

no

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 50% in PEG 300. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no.

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

50% in PEG 300

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

no

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 50% in PEG 300. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no.

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50% in PEG 300

No. with + reactions:

0

Total no. in group:

9

Clinical observations:

No

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50% in PEG 300. No with. + reactions: 0.0. Total no. in groups: 9.0. Clinical observations: No.

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50% in PEG 300

No. with + reactions:

0

Total no. in group:

9

Clinical observations:

No

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50% in PEG 300. No with. + reactions: 0.0. Total no. in groups: 9.0. Clinical observations: No.

Results 5

Reading:

1st reading

Group:

positive control

Remarks on result:

positive indication of skin sensitisation

Results 6

Reading:

2nd reading

Group:

positive control

Remarks on result:

positive indication of skin sensitisation

Any other information on results incl. tables

   Viability / Mortality / Macroscopic Findings

One animal (no. 41) of the test group had to be euthanized on test day 10 (i.e. day of removal of the epidermal induction dressing) due to a prolapsed anus, which was concomitant with blood loss. Necropsy was not performed as the finding was visible.

No necropsies were performed on all surviving animal

 Clinical Signs

No clinical signs were observed throughout the entire observation period.

 

 Body weights

 The body weight of the animals was within the range commonly recorded for animals of this strain and age.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

Based on the above mentioned findings in an adjuvant sensitisation test (M&K-test) in guinea pigs and in accordance to Regulation (EC) No 1272/2008, Sodium Sulphate does not have to be classified as a skin sensitizer.

Executive summary:

In order to assess the cutaneous allergenic potential of Sodium Sulphate, the Maximization-Test was performed in 15 (10 test and 5 control) male albino Dunkin Hartley guinea pigs, in accordance with OECD Guideline No. 406 and the Commission Regulation (EC) No 440/2008, B.6.

 

The intradermal induction of sensitisation in the test group was performed in the nuchal region with a 25% dilution of the test item in PEG 300 and in an emulsion of Freund's Complete Adjuvant (FCA)/physiological saline. The epidermal induction of sensitisation was conducted for 48 hours under occlusion with the test item at 75% in PEG 300 one week after the intradermal induction. The animals of the control group were intradermally induced with PEG 300 and FCA/physiological saline and epidermally induced with PEG 300 under occlusion.

 

Two weeks after epidermal induction the test and control animals were challenged by epidermal application of the test item at 50% in PEG 300 and PEG 300 alone under occlusive dressing.

 

Cutaneous reactions were evaluated at 24 and 48 hours after removal of the dressing.

	After 24 hours				After 48 hours
	Positive / Total				Positive / Total
	% Positive of Total				% Positive of Total
Control Group
Sodium Sulphate, 50% in PEG 300		0 / 5				0 / 5
(left flank)		0				0
PEG 300 only		0 / 5				0 / 5
(right flank)		0				0
Test Group
Sodium Sulphate, 50% in PEG 300		0 / 9*				0 / 9*
(left flank)		0				0
PEG 300 only		0 / 9*				0 / 9*
(right flank)		0				0

*One animal of the test group had to be euthanized due to a prolapsed anus, which was concomitant with blood loss (not treatment related).

No intercurrentdeaths occurred during the course of the study with the exception of oneanimal of the test group, which had to be euthanized on test day 10 (i.e. day of removal of the epidermal induction dressing)) due to a prolapsed anus, which was concomitant with blood loss. Necropsy was not performedas thefinding was visible.

No toxic signs were evident in the surviving guinea pigs of the control or test group.

 

No local skin effects were observed in the surviving guinea pigs of the control or test group.

 

The control and test animals of the main test were sacrificed at the end of the observation period by intraperitoneal injection of pentobarbitone at a dose of 2.0 mL/kg of 162 mg sodium pentobarbitone/mL and discarded. The intradermal and epidermal pretest animals were sacrificed as described above at the treatment start of the main test. Based on the above mentioned findings in an adjuvant sensitisation test (M&K-test) in guinea pigs and in accordance to Regulation (EC) No 1272/2008, Sodium Sulphate does not have to be classified as a skin sensitizer.