dipropyl cyclohexane-1,2-dicarboxylate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 August 2015- 8th September 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

1,2-dipropyl cyclohexane-1,2-dicarboxylate

EC Number:

805-172-8

Cas Number:

65646-25-5

Molecular formula:

C14 H24 O4

IUPAC Name:

1,2-dipropyl cyclohexane-1,2-dicarboxylate

Test material form:

other: Liquid

Details on test material:

Identification: CHP
Alternative name: DNPH-100
Chemical name: Cyclohexane-1,2-dicarboxylic n-propyl ester
CAS number: 65646-25-5
Intended use: Chemical process regulator
Appearance: Colourless liquid
Storage conditions: Room temperature in the dark
Supplier: Sponsor
Lot number: ST111209
Expiry date: End of December 2016
Purity/composition: 98.6%
Sample receipt: 31 March 2015

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source:Harlan Netherlands Ltd.
- Age at study initiation: approximately eight to twelve weeks of age
- Weight at study initiation: The mice were in the weight range 17.4 to 21.3 g
- Housing:They were housed two animals per cage, in solid bottomed polycarbonate cages with a stainless steel mesh lid. Each cage contained a quantity of autoclaved wood flake bedding, additionally Nestlets and a plastic shelter were included for environmental enrichment.
- Diet (e.g. ad libitum): Standard rodent diet Harlan Teklad 2014C Diet. This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent
- Water (e.g. ad libitum): Potable water taken from the public supply was freely available via polycarbonate bottles fitted with sipper tubes.
- Acclimation period: They were acclimatised to the experimental environment for at least 5 days prior to the start of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23°C
- Humidity (%): 40 to 70%
- Air changes (per hr): The animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated.
- Photoperiod (hrs dark / hrs light): Artificial lighting was controlled to give a cycle of 12 hours continuous light and 12 hours continuous dark per 24 hours.

IN-LIFE DATES: From: Day 1 To: Day 6

Study design: in vivo (non-LLNA)

Positive control substance(s):

yes

Remarks:

Hexyl cinnamic aldehyde

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

25, 50% v/v in AOO and the test substance as supplied.
The positive control group received 25% v/v hexylcinnamic aldehyde in AOO based on previous experience at this laboratory.

No. of animals per dose:

Preliminary investigation-Two female mice (per concentration) received a daily application of 25 micro litres for three consecutive days
Main phase- Groups of four mice were treated at one of three concentrations of the test substance. Mice received a daily application of 25 micro litres for three consecutive days. Further groups of four mice were sham dosed, received the vehicle alone or the positive control substance in the same manner. An additional sham control group was added to the main study and consisted of four mice.

In the main phase of the study, five days following the first topical application of test substance (Day 6) all mice were injected via the tail vein with 250 micro litres of phosphate buffered saline containing 3H-methyl Thymidinea (3HTdR: 80 microCi/mL) giving a nominal 20 microCi to each mouse.

Details on study design:

RANGE FINDING TESTS:
- Compound solubility:
- Irritation:
- Lymph node proliferation response:

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Assessment of Skin Sensitization Potential using the Local Lymph Node Assay in the Mouse (Pooled treatment group approach)
- Criteria used to consider a positive response: The test substance is regarded as a sensitizer if at least one concentration of the chemical has a SI of three or more.

TREATMENT PREPARATION AND ADMINISTRATION:
Preliminary investigation-The test substance was applied to the dorsal surface of each ear using an automatic micropipette and was spread over the entire dorsal surface of the ear using the tip of the pipette.
Main phase- The test
substance was applied to the dorsal surface of each ear using an automatic micropipette and
was spread over the entire dorsal surface of the ear using the tip of the pipette. Further groups of four mice were sham dosed, received the vehicle alone or the positive control substance in the same manner.
Sham control group-At dosing, the tip of a pipette was passed over the dorsum of the mouse ears, but no vehicle administered.
Administration of 3H-methyl Thymidine- The injection into the tail vein was carried out using a plastic syringe and needle after the mouse had been heated in a warming chamber.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:

The SI for the positive control substance hexyl cinnamic aldehyde (HCA), was 6.3 which demonstrates the validity of this study.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Remarks:

As supplied

Value:

5.5

Remarks on result:

other: The SI (test/control ratios) obtained for 25, 50% v/v and as supplied were 2.1, 1.1 and 5.5 respectively.

Results 2

Key result

Parameter:

other: disintegrations per minute (DPM)

Remarks:

As supplied

Value:

3 429.6

Remarks on result:

other: Please see table 1 in overall remarks and attachments.

Results 3

Key result

Parameter:

other: dpm/node

Remarks:

As supplied

Value:

857.4

Remarks on result:

other: Please see table 2 in overall remarks and attachments.

Any other information on results incl. tables

Results -Preliminary investigation-Mortality and clinical signs-No signs of ill health or toxicity were observed during this study. Wet/greasy fur on the head was noted in both groups following each dosing occasion, this was related to unoccluded dermal administration of a liquid formulation/vehicle and not an effect of the test substance.

Dermal reactions-No erythema was observed on the ears of either mouse on Days 1 to 6.

Measurement of ear thickness-There was no evidence of an effect of treatment on ear thickness.

Body weight-There was no indication of an overt effect of treatment on body weight gain. On the basis of the results from the preliminary investigation, CHP as supplied was considered a suitable high concentration for administration in the main phase of the study.

Main phase -Mortality and clinical signs-There were no deaths and no signs of ill health or toxicity were observed during this study. Wet/greasy fur on the head was noted in all groups (except the sham dosed group) following each dosing occasion, this was related to unoccluded dermal administration of a liquid formulation/vehicle and not an effect of the test substance.

Dermal reactions-No signs of dermal irritation were seen on the ear during the study.

Body weight-There was no indication of an effect of treatment on body weight gain. Envigo: LBM0091 19 A loss in body weight was noted for four mice (Nos. A9, A20, A25and A27) over the study period, however, a small loss in body weight is not uncommon in young laboratory mice and is not considered to be an effect of treatment.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CHP is regarded as a potential skin sensitizer.

Executive summary:

Summary

The study was performed to assess the skin sensitization potential of CHP using the local lymph node assay (LLNA).

Preliminary investigations were performed at 50% v/v and the test substance as supplied with 2 mice per concentration to establish the highest concentration of test substance which did not lead to systemic toxicity or excessive local irritation. The results of preliminary investigations indicated that the test substance as supplied would be a suitable high concentration for use on the main study.

The study comprised three treated groups, each comprising four female mice receiving CHP at concentrations of 25, 50% v/v or as supplied. Similarly constituted groups were either sham dosed, received the vehicle 4:1 v/v acetone:olive oil (AOO) or positive control substance (25% v/v hexyl cinnamic aldehyde). The mice were treated by daily application of 25 µL of the appropriate concentration or control (vehicle or positive), to the dorsal surface of both ears for three consecutive days. The proliferative response of the lymph node cells (LNC) from the draining auricular lymph nodes was assessed five days following the initial application, by measurement of the incorporation of 3H-methyl Thymidine (3HTdR) by ß-scintillation counting of LNC suspensions. The response was expressed as radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of 3HTdR incorporation into LNC of test nodes relative to that recorded for control nodes (test/control ratio), termed as Stimulation Index (SI). The test substance is regarded as a sensitizer if at least one concentration of the chemical has a SI of three or more.

Results

The SI results obtained for 25, 50% v/v and as supplied were 2.1, 1.1 and 5.5 respectively which indicates that CHP showed the potential to induce skin sensitization. The SI for the positive control substance hexyl cinnamic aldehyde was 6.3, which demonstrates the validity of this study.

Conclusion

CHP is regarded as a potential skin sensitizer.