Registration Dossier

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:
2003-12-01 to 2004-05-06
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2002
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
draft June 2001
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
March 2003
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Specific details on test material used for the study:
Vehicle
Acetone/Olive oil (4:1 v/v)
Rationale
The vehicle was selected as suitable vehicle at request of the sponsor and based on trial formulations perlormed at NOTOX.
Preparation
The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. No adjustment was made for specific gravity of the vehicle. Homogeneity was obtained to visually acceptable levels.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Species
Mouse, CBA strain, inbred, SPF-Quality.
Recognised 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
30 females (six groups of five females each group).
(nulliparous and non-pregnant).

Age and bodyweight
Young adult animals (approx. 10 weeks old) were selected. Body weight variation for the majority of animals was within +/- 20% of the sex mean. The body weights of six animals were outside the range of 21 ± 4 grams.

Identification
Tailmark.

Control animals
The results of the vehicle control animals in NOTOX Project 398058 were used as reference for the initially treated animals of this study and the results of the vehicle control animals, treated in NOTOX Project 401028 were used as reference for the additional treated animals of this study.
The vehicle control animals were treated using the same vehicle, using the same procedures and within the same time frame as this study. The control animals used for the initially treated animals were 14 weeks old and were considered suitable to be used as controls for this study.

Reliability check
The results of a reliability test performed not more than 6 months previously or 2 months afterwards are summarised in the Appendix. Similar procedures were used in the reliability test and in this study.

Animal husbandry
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 ± 3.0°C (actual range: 17.9 - 21.6°C), a relative humidity of 30-70% (actual range: 29 - 70%) and 12 hours artificial fluorescent light and 12 hours darkness per day.

Accommodation
Individual housing in labelled Macrolon cages (type I; height 12.5 cm) containing purified sawdust as bedding material (Woody-Clean type 3/4; Tecnilab-BMI BV, Someren , The Netherlands). Certificates of analysis were examined and then retained in the NOTOX archives. The acclimatisation period was at least 5 days before the start of treatment under laboratory conditions. Animals were group housed in polycarbonate cages (Macrolon II type; height 15 cm) during the acclimatisation period.

Diet
Free access to standard pelleted laboratory animal diet (from Altromin (code VRF 1), Lage, Germany). Certificates of analysis were examined and then retained in the NOTOX archives.

Water
Free access to tap-water. Certificates of quarterly analysis were examined and then retained in the NOTOX archives.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0.25 - 1 - 2.5 - 5 - 25 - 50%
No. of animals per dose:
5
Details on study design:
Preliminary irritation study
A preliminary irritation study was conducted in order to select the highest test substance concentration to be used in the main study. In principle, this concentration should be well tolerated systemically by the animals and may give moderate irritation (grade 3) at the highest.
A series of four test substance concentrations were tested, the highest concentration being the maximum concentration that could technically be applied. The starting- and subsequent concentrations were taken from the series: 100% (undiluted), 50%, 25%, 10%, 5%,2.5%, 1% and if needed further lower concentrations using the same steps. The test system, procedures and techniques were identical to those used during days 1 to 3 of the main study unless otherwise specified.
Four young adult animals were selected (5-14 weeks old). Each animal was treated with one concentration on two consecutive days. Approximately 4 hours after the last exposure, the skin was cleaned of residual test substance with water and the irritation was assessed. No necropsy was performed and no bodyweights were determined after termination.

Main study
Initially, three groups of five animals were treated with three test substance concentrations respectively.
Based on the results, three additional groups were treated with three lower concentrations.

INDUCTION
GROUP* INDUCTION
5 (21-25) Experimental Lowest test substance concentration: 5%
6 (26-30) Experimental Intermediate test substance concentration: 25%
7 (31-35) Experimental HiQhest test substance concentration: 50%
*. five females each group, animal numbers between brackets Allocation additional groups

GROUP* INDUCTION
8 (36-40) Experimental test substance concentration: 0.25%
9 (41-45) Experimental test substance concentration: 1.0%
10 (46-50) Experimental test substance concentration: 2.5%
*. five females each group, animal numbers between brackets

INDUCTION - Days 1, 2 and 3
Experimental animals:
The dorsum surface of both ears was epidermally treated (25 µg/ear) with the test substance concentration, approximately the same time each day.
TREATMENT - Day 6:
All animals:
Each animal was injected via the tail vein with 0.25 ml of sterile phosphate buffered saline (PBS) containing 20 !-µCi of 3H-methyl thymidine (Amersham Pharmacia Biotech, NOTOX Substance 105624).
After approximately five hours, all animals were killed by intra peritoneal injection with an overdose of pentobarbital. The draining (auricular) Iymph node of each ear was excised.
The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes were recorded. The nodes were pooled for each animal in 3 ml PBS.
6.6. Tissue processing for radioactivity
A single cell suspension of Iymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter 125 µm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4°C. The DNA was precipitated with 3 ml 5% trichloroacetic acid (TCA) at 4° C for approximately 18 hours.
Precipitates were recovered by centrifugation at 200g for 10 minutes, resuspended in 1 ml TCA and transferred to 10 ml of Ultima Gold (Packard) as the scintillation fluid.
6.7. Radioactivity measurements
All radioactive measurements were performed using a Packard scintillation counter (1900TR).
Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever comes first. The Packard 1900TR was programmed to automatically subtract background and convert CPM to DPM.

Observation
Mortality/Viability
Twice daily.
Toxicity
At least once daily.
Body weights
On days 1 (pre-treatment) and 6.
Irritation
On day 3 (3-4 hours after treatment), the skin reactions were assessed. If possible, skin reactions were graded according to the following numerical scoring system. Furthermore, descriptions of all other (Iocal) effects were recorded.

Grading Irritation Reactions:
Erythema and eschar formation:
No erythema ...................................................................................................................................... 0
Slight erythema (barely perceptible) ................................................................................................... 1
Well-defined erythema ....................................................................................................................... 2
Moderate erythema .......................................................................................................................... 3
Severe erythema (beet redness) 10 slight eschar formation (injuries in depth) ..................................... .4
Oedema formation:
Nooedema ........................................................................................................................................ 0
Slight oedema (barely perceptible) ...................................................................................................... 1
Well-defined oedema (edges of area well-defined by definite raising) .................................................. 2
Moderate oedema (raised approximately 1 millimeter) .......................................................................... 3
Severe oedema (raised more than 1 millimeter and extending beyond the area of exposure) ................. 4
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 March 2004, females ofthe CBA mouse strain (from Charles River France, L'Arbresle Cedex, France) were checked for the sensitivity to ALPHA-HEXYLCINNAMICALDEHYDE, TECH. 85%.
The females were apprax. 10 weeks old at commencement of the study. The study was based on the OECD Guideline No. 429, the EC Directive 67/548/EC, Part 8.42 and on the method described by ICCVAM (NIH publication; No 99-4494, February 1999). ALPHAHEXYLCINNAMICALDEHYDE, TECH. 85% (CAS no. 101-86-0) was fabricated under lot no. 10021 HF (Aldrich Chemicals Co., Germany). Concentrations used for this study were 5, 10 and 25% in Acetone:Olive oil (4:1).

CONCLUSION
The SI values calculated for HCA concentrations 5, 10 and 25% were 1.2, 2.7 and 16.8 respectively.
An EC3 value of 10.3% was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range of 2 and 20%.
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:
EC3
Value:
3.4
Parameter:
SI
Value:
1.4
Test group / Remarks:
8
Parameter:
SI
Value:
2.5
Test group / Remarks:
9
Parameter:
SI
Value:
2.8
Test group / Remarks:
10
Parameter:
SI
Value:
18.4
Test group / Remarks:
5
Parameter:
SI
Value:
16.7
Test group / Remarks:
6
Parameter:
SI
Value:
6.1
Test group / Remarks:
7

Any other information on results incl. tables

PRELIMINARY IRRITATION 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 Table 1.

Based on the results, the test highest test substance concentration selected for the main study was a 50% concentration.

MAIN STUDY

Induction phase (Table 2)

The skin effects seen after the third epidermal exposure are presented in the table.

Macroscopy (Table 2)

The sizes of the majority of nodes were considered to be normal, except for a few nodes among the experimental animals. No other macroscopic abnormalities of the nodes were noted.

Body Weights (Table 2)

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The slight body weight loss, noted in some animals, was considered not toxicologically significant.

Radioactivity Measurements (Table 3)

Mean DPM/animal values for the experimental groups treated with test substance concentrations 5, 25 and 50% were 2784,2522 and 921 respectively. The mean DPM/animal value for the vehicle contra I group was 151 (NOTOX Project 398058).

In order to clarify the dose response relationship, additional groups of animals were treated with test substance concentrations 0.25, 1 and 2.5% were 363,656 and 745 respectively. The mean DPM/animal value for the vehicle control group was 262 (NOTOX Project 401028).

ToxicityIMortality

No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

PRELIMINARY IRRITATION STUDY

Table 1: Grading of Skin Reactions after Epidermal Exposure

Day1                                                   dorsal surface ear (Day2)

animal        Body            cone.%                         left                                  right

number

weight (g)

 

erythema

oedema

erythema

oedema

 

1

 

23

 

5

 

0

 

0

 

0

 

0

2

24

10

0

0

0

0

3

22

25

0

0

0

0

4

23

50

0

0

0

0

Note: The 50% concentration was applied using a spatula, instead of using a pipette.

MAIN STUDY

 

Table 2: Skin reactions after 3rd induction, Body Weights and Relative Size Nodes

 

 

Da;t1

Da;t3

 

Da;t6

group

an

 

bw

skin grading dorsal surface ear

bw

size nodes

treatment

no

 

(g)

left                         right

(g)

left        right

                                                                    erythema     oedema      erythema       oedema                                                                 

 

1

1

22

0

0

0

0

21

n

n

Vehicle•

2

26

0

0

0

0

25

n

n

Control

3

23

0

0

0

0

22

n

n

 

4

23

0

0

0

0

20

n

n

 

5

26

0

0

0

0

23

n

n

5

21

23

0

0

0

0

22

n

n

5% test

22

22

0

0

0

0

23

n

n

substance

23

17

0

0

0

0

18

n

n

 

24

20

0

0

0

0

22

n

n

 

25

23

0

0

0

0

23

n

n

6

26

19

0

0

0

0

19

n

n

25% test

27

24

0

0

0

0

23

+

+

substance

28

22

0

0

0

0

22

n

n

 

29

22

0

0

0

0

23

+

+

 

30

26

0

0

0

0

26

n

n

7

31

24

0

0

0

0

24

n

n

50% test

32

25

0

0

0

0

25

n

n

substance

33

22

0

0

0

0

19

n

n

 

34

22

0

0

0

0

22

n

n

 

35

27

0

0

0

0

27

n

n

Legends: BW =Bodyweight, Relative size nodes(-: reduced,+: enlarged, n: considered to be normal)

 

  Acetone/Olive oil (4:1 v/v) (NOTOX Project398058)

 

Note 1: The 50% concentration was applied using a spatula, instead of using a pipette.

 

Note 2: Body weights of animals 2, 5, 30 and 35 exceeded the weight range of 21 ± 4 grams.

Table 3: Skin reactions after 3rd induction, Body Weights and Relative Size Nodes (cont'd)

 

 

Da 1

Da 3

 

Da 6

group

an

bw

skin grading dorsal surface ear

bw

size nodes

treatment

no

(g)

left                        right

(g)

left        right

                                                                    erythema    oedema     erythema     oedema                                                           

 

ADDITIONAL GROUPS

1

1

22

0

0

0

0

22

n

n

Vehicle*

2

21

0

0

1

0

21

n

n

Control

3

19

0

0

1

0

19

n

n

 

4

22

1

0

0

0

22

n

n

 

5

23

0

0

0

0

23

n

n

8

36

16

0

0

0

0

16

n

n

0.25%

37

18

0

0

0

0

17

n

n

test

38

23

0

0

0

0

23

n

n

substance

39

25

0

0

0

0

25

n

n

 

40

22

0

0

0

0

22

n

n

9

41

17

0

0

0

0

16

++

n

1.0% test

42

18

0

0

0

0

18

n

n

substance

43

21

0

0

0

0

21

n

n

 

44

20

0

0

0

0

19

n

n

 

45

19

0

0

0

0

19

n

n

10

46

18

0

0

0

0

18

n

n

2.5% test

47

20

0

0

0

0

19

n

n

substance

48

16

0

0

0

0

15

n

n

 

49

19

0

0

0

0

18

n

n

 

50

22

0

0

0

0

22

n

++

Legends: BW =Bodyweight, Relative size nodes(-: reduced,+: enlarged, n: considered to be normal)

 

 Acetone/Olive oil {4:1 v/v) (NOTOX Project401028)

 

Note 1: Body weights of animals 36 and 48 were below the weight range of 21±4 grams.

Table 4: Radioactivity measurements {individual animals)

 

group

animal

treatment

Induction

DPM / animal

 

1

 

1

 

vehicle control

 

Acetone/Olive oil (4:1}

 

86

 

2

 

(NOTOX Project 398058)

66

 

3

 

 

284

 

4

 

 

103

 

5

 

 

216

5

21

experimental

5% test substance

2003

 

22

 

 

3407

 

23

 

 

3499

 

24

 

 

1756

 

25

 

 

3257

6

26

experimental

25% test substance

1800

 

27

 

 

2432

 

28

 

 

3227

 

29

 

 

2833

 

30

 

 

2319

7

31

experimental

50% test substance

692

 

32

 

 

1681

 

33

 

 

728

 

34

 

 

169

 

35

 

 

1337

Table 5: Radioactivity measurements (individual animals) (cont'd)

 

group

animal

treatment

Induction

DPM / animal

 

1

 

1

 

vehicle control

 

Acetone/Olive oil (4:1)

 

219

 

2

 

(NOTOX Project 401028)

280

 

3

 

 

243

 

4

 

 

464

 

5

 

 

105

8

36

experimental

0.25% test substance

312

 

37

 

 

251

 

38

 

 

197

 

39

 

 

428

 

40

 

 

625

9

41

experimental

1% test substance

392

 

42

 

 

611

 

43

 

 

708

 

44

 

 

519

 

45

 

 

1050

10

46

experimental

2.5% test substance

820

 

47

 

 

1101

 

48

 

 

38#

 

49

 

 

677

 

50

 

 

383

 

 #.Value rejected. Very low and outside the range.


 

Table 6: Calculation of Stimulation Index (SI)

 

group

treatment

induction

mean

Sl±SD

 

 

 

DPM ±SD

 

8**

 

experimental

 

0.25% test substance

 

363 ± 170

 

1.4 ± 0.7

9**

experimental

1% test substance

656 ± 249

2.5 ± 0.6

10**

experimental

2.5% test substance

745 ± 299

2.8 ± 0.6

5*

experimental

5% test substance

2784 ± 835

18.4 ± 0.7

6*

experimental

25% test substance

2522 ± 540

16.7 ± 0.7

7*

experimental

50% test substance

921±593

6.1 ± 0.9

1**

vehicle control

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

262 ± 130

1.0

1*

vehicle control

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

151±94

1.0

 

 

•. Asterisk indicates the concurrent control used to calculate the SI.


Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The SI values calculated for the substance concentrations 0.25, 1, 2.5, 5, 25 and 50% were 1.4, 2.5,2.8,18.4,16.7 and 6.1 respectively.
These data showed a dose-response and an EC3 value of 3.4% was calculated.
Based on these results and according to the recommendations made in the test guidelines (OECD No.429, EC B.42 and EPA OPPTS 870.2600), naphthalene-1,5-diol should be regarded as a skin sensitiser.
Based on these results and according to the:
- OECD Harmonized Integrated Hazard Classification System for Human Health and Enviranmental Effects of Chemical Substances (OECD, 1998), naphthalene-1,5-diol should be classified as contact sensitiser.
- EC criteria for classification and labelling requirements for dangerous substances and preparations (Council Directive 67/548/EEC), naphthalene-1,5-diol should be labelled as: may cause sensitisation by skin contact (R 43).
Executive summary:

Assessment for Contact Hypersensitivity to naphthalene-1,5-diol 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), Paris Cedex; EC, Council Directive 67/548/EEC, Annex IV C, B.42 (Draft) (2001); Environmental Protection Agency (EPA): Health Effects Test Guidelines OPPTS 870.2600. "Skin Sensitisation" 2003.

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

In the main study, three groups of five experimental animals were epidermally exposed to a 5%, 25% and 50% concentration respectively on three consecutive days. Five vehicle control animals were similarly treated, but with vehicle alone (Acetone/Olive oil (4:1 v/v)(NOTOX Project 398058).

Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) Iymph nodes were excised.

After precipitating the DNA of the Iymph node cells, radioactivity measurements were done. Based on the results, additional groups were treated at 0.25, 1 and 2.5%. Five vehicle control animals were similarly treated, but with vehicle atone (Acetone/Olive oil (4:1 v/v)(NOTOX Project 401028).

The sizes of the majority of nodes were considered to be normal, except for a few nodes among the experimental animals. No other macroscopic abnormalities of the nodes were noted.

Mean DPM/animal values for the experimental groups treated with test substance concentrations 5, 25 and 50% were 2784, 2522 and 921 respectively. The mean DPM/animal value for the vehicle control group was 151.

In order to clarify the dose response relationship, additional groups of animals were treated with test substance concentrations 0.25, 1 and 2.5% were 363,656 and 745 respectively. The mean DPM/animal value for the vehicle control group was 262.

The SI values calculated for the substance concentrations 0.25, 1, 2.5, 5, 25 and 50% were 1.4, 2.5,2.8,18.4,16.7 and 6.1 respectively.

These data showed a dose-response and an EC3 value of 3.4% was calculated.

Based on these results and according to the recommendations made in the test guidelines (OECD No.429, EC B.42 and EPA OPPTS 870.2600), naphthalene-1,5-diol should be regarded as skinsensitiser.

Based on these results and according to the:

- OECD Harmonized Integrated Hazard Classification System for Human Health and Environmental Effects of Chemical Substances (OECD, 1998), naphthalene-1,5-diol should be classified as contact sensitiser.

- EC criteria for classification and labelling requirements for dangerous substances and preparations (Council Directive 67/548/EEC), naphthalene-1,5-diol should be labelled as:

may cause sensitisation by skin contact (R 43).