Reaction mass of 2-hydroxyisopropyl 2-hydroxypropyl bicarbamate and 2-hydroxypropyl carbazate and propylene carbonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

- modification: in addition, measurements of ear swelling and ear weight were done to discriminate the irritating potential from the sensitizing potential of the test substance (Integrated Model for the Differentiation of Skin reactions (IMDS))

Principles of method if other than guideline:

Modified LLNA (IMDS; Integrated Model for the Differentiation of Skin Reactions). Modifications are authorised in the OECD TG 429 and in the Note for Guidance SWP/2145/00 of the CPMP (2001). Information on validation of IMDS and scientific justification is given in: Vohr HW et al., Arch. Toxicol., 73, 501-509 (2000); Ehling G et al., Toxicology 212, 60-68 and 69-79 (2005).

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:

Reaction mass of 2-hydroxyisopropyl 2-hydroxypropyl bicarbamate and 2-hydroxypropyl carbazate and propylene carbonate

EC Number:

906-521-8

Cas Number:

85391-60-2

Molecular formula:

C8H16N2O6

IUPAC Name:

Reaction mass of 2-hydroxyisopropyl 2-hydroxypropyl bicarbamate and 2-hydroxypropyl carbazate and propylene carbonate

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 38
- Purity: 100 %
- Expiration date of the lot/batch: 2003-09-03

In vivo test system

Test animals

Species:

mouse

Strain:

NMRI

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan-Winkelmann GmbH, Borchen, Germany
- Age at study initiation: 8-9 weeks
- Weight at study initiation: 26-33 g
- Housing: 1 animal/cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 40 - 70
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:

other: DAE 433 (40 % dimethylacetamide, 30 % acetone and 30 % ethanol)

Concentration:

0, 3, 10, 30 %

No. of animals per dose:

6

Details on study design:

TREATMENT PREPARATION AND ADMINISTRATION:
The test item in the formulation or the vehicle were applied epicutaneously onto the dorsal part of both ears of the animals. This  treatment was repeated on three consecutive days (d1, d2 and d3). The volume administered was 25µL/ear. The used concentrations were based on the experiences with the test system and the toxic properties of the test substance.
The animals were anaesthetized by inhalation of carbon dioxide and sacrificed one day after the last application (d4). The appropriate organs were then removed. Lymphatic organs (the auricular lymph nodes) were transferred into physiological saline (PBS).
Investigations:
- weight of draining lymph nodes (given as weight index compared to vehicle controls)
- cell counts in draining lymph nodes (given as cell count index compared to vehicle controls)
Stimulation indices were calculated by dividing the absolute weight or number of cell counts of the substance treated lymph nodes by the vehicle treated ones.
- ear swelling (given in 0.01 mm and as index)
- ear weight (given in mg/8 mm diameter punch and as index)
- body weights

Positive control substance(s):

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

Statistics:

When it was statistically reasonable, the values from treated groups were compared with those from the control group by the Mann-Whitney or the Wilcoxon significance test (Rank Sum Test or One Way ANOVA or Kruskal-Wallis ANOVA) at significance levels of 5 % (one-tailed for LLNA/IMDS or PNLA (larger)) . Outlying values in the LN weights were eliminated at a probability level of 99 % by Nalimov's method. In addition, for the LLNA/IMDS the smallest significant differences in the means were calculated by Scheffels method, which according to Sachs can be used for both equal and unequal sample sizes.

Results and discussion

Positive control results:

Alpha hexyl cinnamic aldehyde, checked in regular intervals, showed a clear sensitizing potential in the local lymph node assay (IMDS).

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Remarks:

cell count index (%)

Value:

0.88

Test group / Remarks:

3 % test item

Results 2

Key result

Parameter:

SI

Remarks:

cell count index (%)

Value:

1.07

Test group / Remarks:

10 % test item

Results 3

Key result

Parameter:

SI

Remarks:

cell count index (%)

Value:

0.76

Test group / Remarks:

30 % test item

Any other information on results incl. tables

Table 1: Summary of the LLNA/IMDS results (means of 6 animals per group)

Parameter investigated	Vehicle control	Dose 3 %	Dose 10 %	Dose 30 %
Stimulation index: weight of draining lymph nodes	1.00	0.63 *	0.81	0.72 *
Stimulation index: cell count in draining lymph nodes	1.00	0.88	1.07	0.76
Ear swelling in 0.01 mm on day 4 (index)	19.00 (1.00)	18.83 (0.99)	19.00 (1.00)	18.83 (0.99)
Ear weight in mg / 8 mm diameter punch on day 4 (index)	10.92 (1.00)	10.82 (0.99)	10.70 (0.98)	10.96 (1.00)

* statistically significant decrease (p ≤ 0.05)

The mice did not show increases in stimulation indices for cell counts or for weights of the draining lymph nodes after application of the test item. The “positive level” indicating sensitizing potential, which is 1.35 for the cell count index, was never reached or exceeded in any dose group.

The “positive level” of ear swelling indicating irritating potential, which is 2 x 10-2 mm increase, i.e. about 10 % of the control values, was not reached or exceeded in any dose group. No substance specific effects were determined for ear weights either.

Slight significant decreases compared to vehicle treated animals regarding the weight of the draining lymph nodes were detected in the lowest and highest dose group. These decreases are in the normal range of variance for these parameters and are such of no biological relevance.

The body weights of the animals were not affected by any treatment.

Applicant's summary and conclusion

Interpretation of results:

other: not sensitising

Executive summary:

KGD 1409 was investigated in the modified local lymph node assay (LLNA-IMDS) on female mice according to OECD TG 429. Concentrations of 0 (vehicle control), 3, 10 and 30 % formulated in DAE 433 (40 % dimethylacetamide, 30 % acetone and 30 % ethanol) were tested. The results show that the test item has neither an irritating nor a sensitising potential in mice after dermal application.