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Description of key information

A positive result is reported in a Maximisation assay performed with acrylonitrile

Key value for chemical safety assessment

Skin sensitisation

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Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4 April to 12 May 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Study is a data requirement.
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The study was performed prior to the introduction of the LLNA test guideline.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
The animals were female SPF Dunkin-Hartley guinea pigs, obtained from Charles River Wiga (Germany), weighing 303-423 g. They were acclimatised for at least 5 days, and were approximately 11 weeks old at the start of treatment.
The guinea pigs were housed in pairs in cages with wire-mesh floors. The animal room was air-conditioned at 7.5-15 air changes per hour, a temperature of 21±3°C, relative humidity 30-70% and a 12 hour light/dark cycle. Individuals were identified by tattoo.
The animals were given free access to standard guinea pig diet including 1600 mg/kg ascorbic acid: LC 23-B. Hope Farms (The Netherlands). Hay was also provided one a week (Broekman Institute, The Netherlands). Tap water diluted with decalcified water was provided ad libitum.
Route:
intradermal and epicutaneous
Vehicle:
physiological saline
Remarks:
and milli-R0 water
Concentration / amount:
Sensitisation was induced by an intradermal injection of 2.5% acrylonitrile and an epidermal application of 2% acrylonitrile 7 days later. Animals were challenged at concentrations of 0.2, 0.5 and 1.0%.
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Remarks:
and milli-R0 water
Concentration / amount:
Sensitisation was induced by an intradermal injection of 2.5% acrylonitrile and an epidermal application of 2% acrylonitrile 7 days later. Animals were challenged at concentrations of 0.2, 0.5 and 1.0%.
No. of animals per dose:
10 controls and 20 test subjects.
Details on study design:
Ten females were allocated to the control group and 20 females were allocated to the experimental group, an additional 5 animals were used for the primary irritation test one week prior to the main study.

Primary irritation test: four intradermal injections (0.1 ml/site) were made into the clipped shoulder region of one guinea pig at a concentration of 2.5% (w/w) of the test material in milli-R0 water. The resulting dermal reactions (erythema/necrosis and diameter) were evaluated 24 and 48 hours later. The same animall was also treated epicutaneously on the shaved left flank with 0.5 ml of a 10% concentration of the test substance in milli-R0 water using a Metalline patch mounted on micropore tape. The patch was held in place with Coban elastic bandage for 24 hours, and the skin assessed for erythema at 24 and 48 hours after patch removal. A further four animals were shaved on the left flank and exposed for 24 hours to concentrations of 10%, 5%, 2.5% and 1% (w/w) in milli-R0 water (0.05 ml/concentration). The substance was held in place by mean of an occlusive Squar chamber mounte don micropore tape and fixed in place with elastic bandage. The test sites were assessed for erythema and oedema 24 and 48 hours after removal of the dressings.

Main study - Induction
Intradermal injections: On day 1 an area of the dorsal skin from the scapular region was clipped free of hair. Three pairs of intradermal injections were made at the border of a 2x4 cm area as follows: the test substance diluted to 2.5% (w/w) with physiological saline; Freund's Complete Adjuvant (FCA), 50:50 with distilled water for injection; the test substance at 5% (w/w) in physiological saline, emulsified in a 50:50 mixture of FCA.
Epidermal applications: 7 days after the injections, the sacpular area was clipped again, and a 2x4 cm patch of Metalline mounted on Micropore tape was treated with 0.5 ml of the test substance, 2% (w/w) in milli-R0 water was placed over the injection sites. The patch was held in place with elastic bandage for 48 hours. Controls were treated as above but without the test article.
Reaction siites were assessed for erythema and oedema immediately after dressing removal.

Main study - Challenge
Test and control guinea pigs were challenged two weeks after the epidermal induction application. Hair was shaved for a 5x5 cm area on the left flank. The following four concentrations were applied using Square chambers attached to Micropore tape: 1% in milli-R0 water, 0.5% in milli-R0 water, 0.2% in milli-R0 water, milli-R0 water. 0.05 ml of each concentration and the vehicle was placed into a Square chamber, placed onto the flank of the animal and held in palce with Micropore tape and elastic bandage. Dressings and residual test material were removed after 24 hours. The sites were assessed for redness and swelling 24 and 48 hours after removal of the dressings. The test sites were shaved with an electric razor after the first reading.

Animals were observed for mortality/vitality and signs of toxicity once daily, body weights were recorded during acclimatisation and at study termination.
Challenge controls:
Yes - see above
Positive control substance(s):
yes
Remarks:
carried out in December 1988 with formaldehyde
Positive control results:
A 90% sensitisation rate was observed with 0.5% formaldehyde (positive control)
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
1.0 %
No. with + reactions:
19
Total no. in group:
20
Clinical observations:
Redness and swelling, some scaliness
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 0.5%. No with. + reactions: 19.0. Total no. in groups: 20.0.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
0.5 %
No. with + reactions:
19
Total no. in group:
20
Clinical observations:
Redness and swelling, some scaliness
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 1.0%. No with. + reactions: 19.0. Total no. in groups: 20.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
0.2 %
No. with + reactions:
16
Total no. in group:
20
Clinical observations:
mid but confluent swelling to Redness with swelling
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 0.2%. No with. + reactions: 16.0. Total no. in groups: 20.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
0
No. with + reactions:
1
Total no. in group:
20
Clinical observations:
Mainly no effects but red spots observed (assumed leakage assumed)
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
0.5 %
No. with + reactions:
18
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
0.25 %
No. with + reactions:
7
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
0.1 %
No. with + reactions:
1
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Some red spots (no swelling)
Remarks on result:
no indication of skin sensitisation
Remarks:
Expected control response

No signs of systemic toxicity were noted during the primary irritation experiments and during the main study. No mortality occurred/ Average body weight gain was similar in treated guinea pigs and controls.

Four animals showed skin irritation after the 48 hours occluded epicutaneous induction exposure. No positive skin reactions were observed in the controls following the challenge exposure.

Nineteen animals showed a positive sensitisation reaction in response to the 1% and 0.5% test concentrations, and 16 animals in response to the 0.2% concentration. Two animals showed a skin reaction to the vehicle alone. It was therefore concluded that leakage of the test material across the Square chambers had cocurred. Another minor reaction to the test material was scaliness.

The resulting sensitisation rate was 95%, indicating that acrylonitrile is an extreme sensitiser according to the Kligman (1966) rating.

Positive skin reactions scored after challenge exposure

 

Acrylonitrile Concentration

1%

0.5%

0.2%

0%

Experimental group – no. animals with positive reaction

19

19

16

1*

Sensitisation rate

95

95

80

5*

Control group – no. animals with positive reaction

0

0

0

0

* it was considered possible that some test material leaked from the other chambers

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The guinea pig maximisation test provided clear evidence of skin sensitisation following acrylonitrile exposure.
Executive summary:

The sensitising properties of acrylonitrile were evaluated in the Guinea Pig Maximisation Test. Under the conditions of the study, acrylonitrile resulted in a 95% sensitisation rate after intracutaneous and epicutaneous application. Acrylonitrile should therefore be classified as a skin sensitiser in Category 1B according to Regulation (EC) 1272/2008.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Acrylonitrile is listed on Annex VI of the CLP Regulation with classification for skin sensitisation (H317: may cause an allergic skin reaction'. In addition, there are also reports of sensitisation in exposed workers.

A guideline-compliant Maximisation assay is also reported (Koopmans & Daamen. 1989). In this study, sensitisation was induced by intradermal injection of 2.5% acrylonitrile and an epidermal application of 2% acrylonitrile. Animals challenged with acrylonitrile concentrations of 0.5% and 1.0% acrylonitrile showed a 95% positive sensitisation rate. Exposure to 0.2% on challenge caused an 80% sensitisation rate.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

No animal data are available; there is no recognised validated test guideline for the investigation of this endpoint. There are no reports, from exposed workers of occupational asthma, which indicates that acrylonitrile does not have the potential to cause respiratory sensitisation.

Justification for classification or non-classification

Acrylonitrile is listed on Annex VI of Regulation 1272/2008/EC with H317 'May cause an allergic skin reaction' . Based on the results of the key maximisation assay, classification in Category 1B is appropriate; however it is notable that there are very few reports of skin sensitisation in exposed workers. No classification for respiratory sensitisation is proposed in the absence of any evidence that acrylonitrile can cause respiratory sensitisation.