Isophthalohydrazide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 September 2016 - 05 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(22 July 2010)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EC No 640/2012, Part B: Skin sensitization: "Local Lymph Node Assay"

Deviations:

no

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:

Isophthalohydrazide

EC Number:

220-425-7

EC Name:

Isophthalohydrazide

Cas Number:

2760-98-7

Molecular formula:

C8H10N4O2

IUPAC Name:

isophthalohydrazide

Test material form:

solid: particulate/powder

Details on test material:

Appearance: White powder
Storage Conditions: room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

(CBA/CaOlaHsd)

Sex:

female

Details on test animals and environmental conditions:

- Source: Envigo RMS B.V., Inc., Horst, The Netherlands
- Age at study initiation: Young adult animals (8 - 12 weeks)
- Weight at study initiation: 15 - 23 g
- Housing: Solid floor polypropylene cages furnished with softwood woodflakes.
- Diet: Free access to pelleted rodent diet (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK)
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 – 25
- Humidity (%): 30 - 70
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

50, 25 or 10% or 0% (w/w)

No. of animals per dose:

5

Details on study design:

Preliminary screening test,
A preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µL of the test item at a concentration of 50% w/w in dimethyl formamide, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on days 1, 2 and 3 and once daily on days 4, 5 and 6 for any clinical signs of toxicity and for ear thickness changes.

MAIN STUDY

ANIMAL ASSIGNMENT
Three groups of five animals were treated with one test substance concentration per group. One group of five animals was treated with vehicle.

TREATMENT PREPARATION AND ADMINISTRATION:
Test substance preparation: Groups of five mice were treated with the test item at concentrations of 50%, 25% or 10% w/w in dimethyl formamide formulated within 2 hours . The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1 , 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.

Rationale for vehicle: This vehicle was chosen as it produced the highest concentration that was suitable for dosing.

Induction - Days 1, 2 and 3; Excision of nodes - Day 6; Tissue processing for radioacitivity - Day 6; Radioactivity measurements - Day 7; Performed according to test guidelines.

3H-Methyl Thymidine Administration
Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 gL of phosphate buffered saline (PBS) containing H-methyl thymidine (3HTdR: 80 HCi/mL, specific activity 2.0 Ci/mmoL) giving a total of 20 HCi to each mouse.

Observations:
Body weights: On day 1 (pre-dose) and day 6 (prior to termination).
Clinical signs and mortality: twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6
Irritation: according to a numerical scoring system (see table 1 below).

Evaluation criteria
The test item will be regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values (SI ≥ 3). Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitizer".

Positive control substance(s):

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

Statistics:

Not performed.

Results and discussion

Positive control results:

A group of five animals was treated with 50 µL (25 µL per ear) of a-Hexylcinnamaldehyde, tech., 85% as a solution in dimethyl formamide at a concentration of 25% v/v gave a stimulation index of 6.08 and was considered positive.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.1

Test group / Remarks:

Concentration test item: 10%

Results 2

Parameter:

SI

Value:

1.5

Test group / Remarks:

Concentration test item: 25%

Results 3

Parameter:

SI

Value:

1.28

Test group / Remarks:

Concentration test item: 50%

Any other information on results incl. tables

Results Pre-screen test:

No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted.

Main study:

Mean DPM/animal values: concentrations 0, 10, 25 and 50% were 2744.39, 3029.80, 4129.74 and 3500.13 DPM, respectively.  The SI values calculated for the test item concentrations 10, 25 and 50% were 1.10, 1.50 and 1.28, respectively.

Other results - main study:

Systemic Toxicity and bodyweight

No mortality or signs of systemic toxicity were noted in the test or control animals.
No abnormalities in body weight were observed

Applicant's summary and conclusion

Interpretation of results:

other: Not skin sensitizing

Conclusions:

In an LLNA skin sensitisation study, performed according to OECD 429 and EC/B42 test guidelines, Isophthalic dihydrazide was considered not to be a skin sensitiser, as the SI appeared not to be ≥ 3 when tested up to 50% (w/w).

Executive summary:

An LLNA skin sensitisation study was performed according to OECD 491 and EC/B42 test guidelines and GLP principles. Based on the results of a pre-screen test, the test concentrations were selected at 10%, 25% and 50% w/w. No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of studies and no abornomalities in body weights was observed over the study period. Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 50% were 3029.80, 4129.74 and 3500.13 DPM, respectively. The mean DPM/animal value for the vehicle control group was 2744.39 DPM. The SI values calculated for the test item concentrations 10, 25 and 50% were 1.10, 1.50 and 1.28, respectively. As the SI appeared not to be ≥ 3 when tested up to 50% w/w, Isophthalic dihydrazide was considered not to be a skin sensitiser.