3-chlorophthalic anhydride

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

1992

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Currently no LLNA study is available for assessment. The Guinea Pig Maximization Test (GPMT) has been carried out as an animal test to predict human sensitization for over a decade and is recommended by international test guidelines such as OECD.

Test material

Test material information

Constituent 1

Reference substance name:

3-chlorophthalic anhydride

EC Number:

204-179-8

EC Name:

3-chlorophthalic anhydride

Cas Number:

117-21-5

Molecular formula:

C8H3ClO3

IUPAC Name:

4-chloro-1,3-dihydro-2-benzofuran-1,3-dione

In vivo test system

Test animals

Species:

guinea pig

Strain:

Hartley

Sex:

male/female

Details on test animals and environmental conditions:

Male and female Hartley guinea pigs were received from Elm Hill Breeding Labs, Inc. They weighed 365.9-466.8 g and were at least 21 days old. They were group-housed upon arrival in stainless steel suspended cages. The animals were acclimated for at least 5 days prior to dosing. Water and feed were provided ad libitum. The temperature and humidity were maintained at 68+/ 5°F and 30-70%, respectively. Room lights were on a 12-hour light/dark cycle.

Study design: in vivo (non-LLNA)

Induction

Route:

intradermal and epicutaneous

Vehicle:

cotton seed oil

Concentration / amount:

- Intradermal: 10%; 0.1 mL injected
- Epicutaneous: 100%; 1 g test substance in 2 mL vehicle

Day(s)/duration:

Intradermal: day 0; Epicutaneous: day 7 with a duration of 48 h

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

cotton seed oil

Concentration / amount:

100%; 1 g test substance in 2 mL vehicle

Day(s)/duration:

Day 21, duration of 24 hours

No. of animals per dose:

- Test group: 5/sex
- Negative control group: 2 males & 3 females
- Intradermal irritation: 1 male & 1 female
- Primary topical irritation: 1 male & 1 female

Details on study design:

A preliminary intradermal irritancy test was conducted, in which 3 CLPA, at concentrations of 100, 50, 10 and 1% in CSO was injected intradermally (0.1 mL/site). It was determined that at 100% and 50% 3 CLPA was severely irritating. At 1% and 10%, 4-CLPA was moderately irritating. In addition, a preliminary topical irritancy test was conducted and 100% 3-CLPA was non-irritating when applied topically. Therefore, 3-CLOX at a concentration of 10% was chosen for the intradermal induction and a concentration of 100% was chosen as the topical induction and challenge doses.

The application sites were prepared by clipping the skin of the test site free of hair. On day 0 and day 7, an approximately 5 x7 cm area over the shoulder region was prepared. On day 21, an approximately 4 x 4 cm square of the flank was prepared.

On Day 0, for the intradermal induction, three pairs of intradermal injections were made so that on either side of the midline there were three injection sites. The dosing solution for each injection is listed below. Injection pairs 1 and 2 were given in close proximity to each other cranially and injection pair 3 was located more caudally. The injection sites were just within the boundaries of 2 x 4 cm.

Test Group
Injection Pair 1: 0.1 mL Freund’s Complete Adjuvant (FCA) 1:1 with CSO
Injection Pair 2: 0.1 mL 3-CLPA at the selected concentration (10% formulation in CSO)
Injection Pair 3: 0.1 mL 3-CLPA 1:1 with FCA

Negative Control Group
Injection Pair 1: 0.1 mL FCA 1:1 with CSO
Injection Pair 2: 0.1 mL CSO (undiluted)
Injection Pair 3: 0.1 mL 50% formulation of CSO 1:1 with FCA

The animals were not treated with sodium lauryl sulfate prior to the topical induction. On Day 7, for the topical induction, the experimental group was dosed with 100% 3-CLPA. 3-CLPA was placed on a 2 x 4 cm piece of filter paper in a thick even layer. The patch was placed on the dorsal surface of the animals, covered by an impermeable sheet, and secured with a non-adhesive bandage which was wound around the torso of the animal. The dressing was left in place for 48 hours. The negative control group was treated in a similar fashion with CSO instead of 3-CLPA.

On Day 21, for the topical challenge, pieces of filter paper measuring 2 x 2 cm were secured ot the flanks for 24 hours, utilizing the same wrapping technique as previously described. For both the test and negative control groups, one patch was placed on the left side with 3-CLPA at 100% and the second patch was placed on the right side with CSO.

Approximately 21 hours after patch removal, the flank skin was cleaned and clipped. Approximately 3 hours later, the first reading of the reactions was performed (Day 23) and the second reading was made on Day 24. For evaluation of skin reactions, the following four-point scale was used:

0 = No reactions
1 = Discrete or patchy erythema
2 = Moderate and confluent erythema
3 = Intense erythema and swelling

Daily observations were made for clinical signs of toxicity. Animals were weighed at the beginning and end of the observation period.

Challenge controls:

1 Negative control group: 2 males/3 females
To ensure the sensitivity and reproducibility of the test procedure, positive controls using a well known contact allergen, i.e. DInitrochlorobenzene, are performed regularly at the testing laboratory.

Positive control substance(s):

yes

Remarks:

Dinitrochlorobenzene

Results and discussion

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

Vehicle

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

Vehicle

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None

Any other information on results incl. tables

All animals gained weight over te course of the study.  No systemic signs of toxicity were observed in any animal over the course of the study.  None of the test animals exhibited any signs of erythema at any of the challenge observation points.  None of the negative control animals exhibited any reaction to the challenge. Moderate to severe erythema and edema was observed at the 100% and 50% concentrations for the Intradermal Primary Irritation animals.  There was well defined erythema and edema at 10% and 1% without any overt signs of Systemic toxicity or tissue destruction.  The Induction animals were dosed with a 10% dosing solution. No erythema or edema was observed at the 100% concentration for the Topical Primary Irritation animals.  The Topical Induction was dosed at 100% concentration.  The Topical Challenge was dosed at 100% concentration.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance was determined to be not skin sensitising.

Executive summary:

Skin sensitisation was determined in a study performed according to OECD 406 and in compliance with GLP criteria. In this study, 5 male and 5 female Hartley guinea pigs were induced intradermal with a 10% solution by a 0.1 mL injection on day 0. On day 7, an epicutaneous induction followed with 100% solution (1 g test substance in 2 mL cotton seed oil), where the test substance was kept under occlusive conditions for 48 hours. On day 21 the animals were challenged by topical application of 100% solution under occlusive conditions for 24 hours. The skin reactions were evaluated 24 and 48 hours after patch removal. Additionally, daily observations were made for clinical signs of toxicity during the test period. Furthermore, animals were weighed at the beginning and end of the observation period. All animals gained weight over the course of the study and no systemic signs of toxicity were observed in any animal over the course of the study. Also, none of the test animals exhibited any signs of erythema at any of the challenge observation points. Based on these results, the test substance is considered to be not skin sensitising.