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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 September 2009 13 October 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to OECD and EU test guidelines and GLP compliant

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Identification Diformal
Chemical name Bis-(2-chlorethyl)-formal; Bis-(2-chloroethoxy)methane
Structure
Molecular formula C5H10Cl2O2
Molecular weight 173.0377
CAS Number 111-91-1
Description Clear yellowish liquid (determined at NOTOX)
Batch 54600-235530
Purity 90.4 %
Test substance storage At room temperature in the dark under nitrogen
Stability under storage conditions Stable
Expiry date 28 February 2010

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Species Mouse, CBA/J strain, inbred, SPF-Quality.
Recognized by the international guidelines as the recommended test system (e.g. OECD, EC, EPA).
Source: Charles River France, L’Arbresle Cedex, France.
Number of animals 20 females (nulliparous and non-pregnant), five females per group.
Age and bodyweight Young adult animals (approx. 10 weeks old) were selected. Body weight variation was within +/- 20% of the sex mean.

Conditions
Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21.0 ± 3.0ºC (actual range: 21.1 – 23.1ºC), a relative humidity of 40-70% (actual range: 37 - 78%) and 12 hours artificial fluorescent light and 12 hours darkness per day.
Cleaning procedures in the room might have caused the temporary fluctuations above the optimal maximum level of 70% for relative humidity. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.

Accommodation
Individual housing in labeled Macrolon cages (MI type; height 12.5 cm) containing sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) was supplied as cage-enrichment.
The paper was removed on Day 1 prior to dosing and was supplied again after scoring of the ears on Day 3.

Acclimatization period
The acclimatization period was at least 5 days before the start of treatment under laboratory conditions. Accommodation was as described above except that the animals were group housed in Macrolon cages (MIII type; height 18 cm).

Diet
Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).

Water
Free access to tap water.

Results of analysis for diet (nutrients and contaminants), sawdust, paper and water were assessed and did not reveal any findings that were considered to have affected the study integrity. All certificates and results of analysis are retained in the NOTOX archives.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0% (vehicle), 5, 10, 25%
No. of animals per dose:
5
Details on study design:
Test substance concentrations selected for the main study were based on the results of a preliminary study.

In the main study, three experimental groups of five female CBA/J mice were treated with test substance concentrations of 5, 10 or 25% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Acetone/Olive oil (4:1 (v/v)).
Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised.
After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of Disintegrations Per Minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
A reliability check is carried out at regular intervals to check the sensitivity of the test system and the reliability of the experimental techniques as used by NOTOX. In this study, performed in August 2009, females of the CBA/J mouse strain (from Charles River France, L’Arbresle Cedex, France) were checked for the sensitivity to Alpha- Hexylcinnamaldehyde, technical grade. The females were approx. 10 weeks old at commencement of the study. The study was based on the OECD Guideline No. 429, EC No 440/2008, Part B.42 and EPA, OPPTS 870.2600 “Skin Sensitization”. Alpha-hexylcinnamicaldehyde, tech. 85% (CAS no. 101-86-0) was fabricated under lot no. 13102MO (Sigma- Aldrich, Steinheim, Germany). Concentrations used for this study were 5, 10 and 25% in Acetone/Olive oil (4:1 (v/v)).

group % test substance 1 mean DPM SI

2 5% 282 1.2
3 10% 535 2.2
4 25% 2066 8.5

1 0% (vehicle) 243 1.0

1. Vehicle: Acetone/Olive oil (4:1 (v/v)).

CONCLUSION

The SI values calculated for the substance concentrations 5, 10 and 25% were 1.2, 2.2 and 8.5 respectively. An EC3 value of 11.9% was calculated using linear interpolation.

The calculated EC3 value was found to be in the acceptable range of 2 and 20%. The results of the 6 monthly HCA reliability checks of the recent years were 13.1, 15.6, 14.1, 13.8, 13.9 and 16.0%. Based on these results it was concluded that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity.

The raw data, protocol and report from this study are kept in the NOTOX archives. The test described above was performed in accordance with NOTOX Standard Operating Procedures and the report was audited by the QA-unit.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The SI values calculated for the substance concentrations 5, 10 and 25% were 1.2, 1.4 and 1.4 respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 5, 10 and 25% were 558, 685 and 662 respectively. The mean DPM/animal value for the vehicle control group was 477.

Any other information on results incl. tables

PRELIMINARY STUDY

The results of the epidermal exposures for the selection of highest test substance concentration to be tested in the main study are described in the table. Mortality occurred at a 100 and 50% test substance concentration. Based on the results, the highest test substance concentration selected for the main study was a 25% concentration.

animal

number

test

substance1

(% w/w)

Day 1

 

Day 3

Body

weight (g)

 

skin reactions dorsal surface ear

Body

weight (g)

 

left

right

 

erythema

oedema

erythema

oedema

 

 

 

 

 

 

 

 

 

12

    50

27

 

n.s.

n.s.

n.s.

n.s.

n.s.

22

   100

29

 

n.s.

n.s.

n.s.

n.s.

n.s.

3

      2.5

19

 

0

0

0

0

18

4

      5

24

 

0

0

0

0

24

5

    10

22

 

1

0

1

0

21

6

    25

19

 

1

0

1

0

19

73

    50

18

 

n.s.

n.s.

n.s.

n.s.

n.s.

 

 

 

 

 

 

 

 

 

 

1. Vehicle: Acetone/Olive oil (4:1 (v/v).

 

2.   Animals sacrificed for ethical reasons on the treatment day after showing ventro-lateral recumbency, labored respiration, piloerection, hunched posture, hypothermia, abnormal gait, uncoordinated movements, ptosis.

 

3.  Animal found dead on Day 3.

 

n.s: Not scored

MAIN STUDY

No oedema was observed in any of the animals examined. The slight irritation of the ears as shown by the animals treated at 10 and 25% was considered not to have a toxicologically significant effect on the activity of the nodes.

 

All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size.

 

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

group

test substance1

(% w/w)

mean

DPM ±

 

SI ±

 

 

 

 

 

 

 

 

2

5%

558

±

18

1.2

±

0.2

3

10%

685

±

83

1.4

±

0.3

4

25%

662

±

108

1.4

±

0.3

 

 

 

 

 

 

 

 

1

0% (vehicle)

477

±

82

1.0

±

0.2

 

 

 

 

 

 

 

 

 

1      Vehicle: Acetone/Olive oil (4:1 (v/v)

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
Based on these results DIFORMAL would not be regarded as skin sensitizer according to the recommendations made in the test guidelines.
Executive summary:

Assessment of Contact Hypersensitivity to DIFORMAL in the Mouse (Local Lymph Node Assay).

 

 

The study was carried out based on the guidelines described in:

OECD, Section 4, Health Effects, No.429 (2002),

EC, No 440/2008; B42: "Skin Sensitization: Local Lymph Node Assay"

EPA, OPPTS 870.2600 (2003) “Skin Sensitization”.

 

 

Test substance concentrations selected for the main study were based on the results of a preliminary study.

 

 

In the main study, three experimental groups of five female/J mice were treated with test substance concentrations of 5, 10 or 25% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Acetone/Olive oil (4:1 (v/v)).

Three days after the last exposure, all animals were injected with3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised.

After precipitating theof the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of Disintegrations Per Minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

 

 

No oedema was observed in any of the animals examined. The slight irritation of the ears as shown by the animals treated at 10 and 25% was considered not to have a toxicologically significant effect on the activity of the nodes.

 

All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size.

 

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

 

Mean DPM/animal values for the experimental groups treated with test substance concentrations 5, 10 and 25% were 558, 685 and 662 respectively. The mean DPM/animal value for the vehicle control group was 477.

 

 


The SI values calculated for the substance concentrations 5, 10 and 25% were 1.2, 1.4 and 1.4 respectively.

 

Since there was no indication that the test substance elicits an SI3 when tested up to 25%, DIFORMAL was considered not to be a skin sensitizer.

 

It was established that the EC3 value(the estimated test substance concentration that will give a SI =3)(if any) exceeds 25%. Since there is no clear dose response relationship, there is no indication that the SI=3 value will be exceeded at higher concentrations.

 

The six monthly reliability check withHexylcinnamaldehyde, indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity.

 

Based on these results DIFORMAL would not be regarded as skin sensitizer according to the recommendations made in the test guidelines.