Calcium nitrite

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 December 1995 - 14 January 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Good quality study, conducted to GLP. The test material was a 34% solution of hydrated calcium nitrite.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

29.12.1992

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

An in vivo skin sensitisation study by the LLNA method does not need to be conducted because adequate data from an existing guinea pig maximisation test are available.

Test material

Test material information

Constituent 1

Reference substance name:

Calcium nitrite

EC Number:

237-424-2

EC Name:

Calcium nitrite

Cas Number:

13780-06-8

Molecular formula:

Ca.2HNO2

IUPAC Name:

calcium nitrite

Constituent 2

Test material form:

liquid

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: D. Hall, Newchurch, Staffordshire, England
- Females (if applicable) nulliparous and non-pregnant: not applicable
- Microbiological status of animals, when known: no data
- Age at study initiation: 4-5 weeks
- Weight at study initiation: 268-327 g
- Housing: Groups of five in suspended metal cages with wire mesh floors
- Diet (e.g. ad libitum): Vitamin C enriched guinea-pig diet FD2 ad libitum
- Water (e.g. ad libitum): drinking water ad libitum
- Acclimation period: 13 days
- Indication of any skin lesions: Animals were described only as "healthy"

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

- IN-LIFE DATES: No data

Study design: in vivo (non-LLNA)

Induction

Route:

intradermal and epicutaneous

Vehicle:

other: water for irrigation (intradermal induction); distilled water (topical induction)

Concentration / amount:

Intradermal induction: 1% DCI v/v
Topical induction: 10% DCI v/v

Day(s)/duration:

Injection on day 1 followed by a 48-hour topical induction 1 week later

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

other: distilled water

Concentration / amount:

2.5 and 5% DCI v/v

Day(s)/duration:

24 hours

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

10 test and 5 control animals.

Details on study design:

RANGE FINDING TESTS: The intradermal and topical irritancy of a range of dilutions of the test substance was investigated to identify where possible a) test substance concentrations that would produce irritation suitable for the induction phase of the main study, and b) a maximum non-irritant topical concentration for the challenge phase. Animals were pretreated with an intradermal injection of Freund's complete adjuvant/water (50:50). For the intradermal injections, 2 guinea pigs were used and 8 concentrations of test material were tested on each animal; 4 topical concentrations were tested on each of 2 additional animals.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 1 intradermal (3 injections) followed 1 week later by 1 topical
- Exposure period: Over the course of a 9-day period
- Test groups: Intradermal injections: 1) Freund’s Complete Adjuvant (FCA) diluted with an equal volume of water for irrigation. 2) The test article, diluted to 1% with water for irrigation. 3) The test article diluted to 1% in a 50:50 (v/v) mixture of FCA and water for irrigation. Topical application: A patch saturated with 0.4 mL of DCI (diluted to 10% v/v in distilled water) was placed on the clipped skin of the test animals for 48 hours. The patch was covered by a length of impermeable plastic adhesive tape, secured with an elastic adhesive bandage wound round the torso of each animal and fixed with impervious plastic adhesive tape.
- Control group: Similarly treated to the test animals with the exception that the test substance was omitted was the intradermal injections and topical application.
- Site: An area of dorsal skin (clipped free of hair prior to both inductions) from the scapular region (40 mm x 60 mm).
- Frequency of applications: Injection at three sites on day 1 and topical application on day 8
- Duration: Topical application was for 48 hours (occluded)
- Concentrations: 1% for intradermal injections (selected since this was the highest concentration that caused irritation but did not adversely affect the animals) and 10% for topical application (selected as this was the highest concentration that produced some irritation but did not adversely affect the animals).

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 2 weeks after topical induction
- Exposure period: 24 hours
- Test groups: Patches (20 mm x 20 mm) saturated with DCI at respective concentrations of 2.5 and 5% v/v were applied to the posterior and anterior sites on the left flank of each animal. The patches were sealed under strips of impermeable plastic adhesive tape, covered with an elastic adhesive bandage wound round the trunk of each animal and secured with impervious plastic adhesive tape.
- Control group: Similarly treated to the test animals (challenge with DCI)
- Site: An area on the left flank of each animal was clipped and shaved.
- Concentrations: 2.5 and 5% for topical challenge (selected as 5% was the highest concentration that did not produce irritation).
- Evaluation (hr after challenge): 24, 48 and 72 hours after removal of the challenge patches.

OTHER: All animals were observed daily for signs of ill health or toxicity. Body weights of individual animals were measured on day 1 and on the final day of observation following the challenge application.

Dermal reactions following the induction and challenge phases of the study were assessed using the following system:
Erythema and eschar formation: No erythema (0); slight erythema (1); well-defined erythema (2); moderate erythema (3); severe erythema (beet redness) to slight eschar formation (injuries in depth) (4).
Oedema formation: No oedema (0); slight oedema (1); well-defined oedema (edges of area well-defined by definite raising) (2); Moderate oedema (raised approximately 1 mm) (3); severe oedema (raised by more than 1 mm and extending beyond the exposure area) (4).
The diameter (mm) of the dermal response at the intradermal injection sites was recorded in the preliminary study only to assist in the choice of concentrations for the main study. Any other lesion not covered by this scoring system was described.

Challenge controls:

Similarly treated as test group

Positive control substance(s):

yes

Remarks:

hexyl cinnamic aldehyde (the sensitivity of the guinea-pig strain is checked periodically at the testing facility using this known sensitiser)

Results and discussion

Positive control results:

A selection of tests using the known sensitiser hexyl cinnamic aldehyde (positive control) were conducted at the testing facility in the years prior to this investigation. All seven studies utilised 10 test animals (with 5 or 10 control animals). In one study, 80% of the animals of the test group had positive skin reactions following treatment with 50% (and as supplied) hexyl cinnamic aldehyde in aqueous solution; a 90% positive response was seen in two studies and a further four studies showed a 100% positive response.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

2.5% DCI v/v

No. with + reactions:

0

Total no. in group:

10

Results 2

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

5% DCI v/v

No. with + reactions:

1

Total no. in group:

10

Clinical observations:

Slight erythema was observed in one animal

Results 3

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

2.5% DCI v/v

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

Necrotic edge was observed in one animal; dryness and sloughing of the epidermis was seen in the same animal and in two others

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

5% DCI v/v

No. with + reactions:

3

Total no. in group:

10

Clinical observations:

Necrotic edge and dryness/sloughing of the epidermis were observed in three and six animals, respectively; three animals showed slight erythema (persisted at the 72-hr time point in only one of these animals)

Results 5

Reading:

other: 3rd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

2.5% DCI v/v

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

Necrotic edge was observed in one animal; dryness and sloughing of the epidermis was seen in another

Results 6

Reading:

other: 3rd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

5% DCI v/v

No. with + reactions:

1

Total no. in group:

10

Clinical observations:

Necrotic edge and dryness/sloughing of the epidermis were observed in three and four animals, respectively; one animal showed slight erythema

Results 7

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

2.5 and 5% DCI v/v

No. with + reactions:

0

Total no. in group:

5

Results 8

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

2.5 and 5% DCI v/v

No. with + reactions:

0

Total no. in group:

5

Results 9

Reading:

other: 3rd reading

Hours after challenge:

72

Group:

negative control

Dose level:

2.5 and 5% DCI v/v

No. with + reactions:

0

Total no. in group:

5

Results 10

Reading:

other: Summary of positive control data (results for seven studies)

Group:

positive control

Dose level:

As supplied (and 50%)

No. with + reactions:

66

Total no. in group:

70

Any other information on results incl. tables

Slight irritation reported at induction with test material.

No signs of ill health or toxicity were reported, and growth was recorded for all guinea-pigs.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In a guideline study, to GLP, calcium nitrite (hydrate) was not sensitising to guinea pigs dermally challenged with the test compound at up to a concentration of 5% following a two-stage induction with 1% by intradermal injection and 10% by topical application.

Executive summary:

The skin sensitisation potential of calcium nitrite (hydrate) was assessed in an EU Method B.6 guinea pig maximisation test (GPMT) using groups of 10 test and 5 control animals. The test material (DCI) was available as a 34% aqueous solution of calcium nitrite.

Male albino guinea pigs were induced, on clipped skin, with a 1% aqueous solution of the test material by intradermal injection (in combination with a preparation of Freund’s complete adjuvant), followed one week later by a second induction by skin application of a 10% aqueous solution of the test substance under a 48-hr occlusive patch. (These concentrations (1 and 10%) were the highest that caused irritation, but not adverse systemic effects, in the preliminary test.) Control animals were similarly treated but without the test substance. Two weeks after the second (dermal) induction, a challenge concentration of 2.5 and 5% of the test material were applied under 24-hr occlusive patches to both test and control animals. (These dermal challenge concentrations were selected following the results of a preliminary study, in which 5% is considered the highest non-irritant concentration). Dermal reactions were evaluated 24, 48 and 72 hours after removal of the challenge patches. No clinical signs of toxicity were reported.

No positive skin sensitisation reactions were observed at any time point following dermal challenge at a concentration of 2.5% test material. One test animal displayed slight transient erythema upon examination 24 hr after removal of the 5% challenge patch, persisting to the 48-hr time point. An additional two animals showed similar effects at this time point, though these observations persisted at the 72-hr time point in only a single animal. Necrotic edge was apparent in three animals at the 48 and 72-hr time points. Calcium nitrite (hydrate) produced no evidence of skin sensitisation in eight of the ten test animals in this GPMT, producing an inconclusive response in one animal and a positive response in another. Overall, the test material is not considered to be a skin sensitiser in this assay.

Based on the results of this study, calcium nitrite does not warrant classification for skin sensitisation, according to EU CLP criteria (EC 1272/2008).