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Administrative data

Description of key information

The substance ‘Naphtha (petroleum), steam-cracked, C8-10 aromatic hydrocarbon fraction, alkylated and oligomerised’ (NAF-AO) [EC no. 701-299-7] (see Chap. 1) was tested for skin sensitisation in two local lymph node assays. Stimulation indices above 3 in both tests clearly indicate that the substance NAF-AO has the potential for skin sensitisation.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Principles of method if other than guideline:
Test material is 'Naphtha (petroleum), steam-cracked, C8-10 aromatic hydrocarbon fraction, alkylated and oligomerised' in form of the technical product Novares L 100.
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
- Name of test material (as cited in study report): Novares L 100; CAS-no. 71302-83-5 (Hydrocarbons, C9- unsaturated, polymerized)
- Composition of test material, percentage of components: see Section 1.2 Composition
- Lot/batch No.: 28738
- Stability under test conditions: no measured data; based on chemical structure assumed to be stable
- Storage condition of test material: room temperature, exclusion of light
Species:
mouse
Strain:
Balb/c
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Breeding farm VELAZ s.r.o., Koleč u Kladna, Czech Republic
- Age at study initiation: 8 - 10 wks
- Weight at study initiation: 17 - 19 g
- Housing: macrolon cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 – 70
- Photoperiod (hrs dark / hrs light): 12 / 12

Vehicle:
other: DAE 433 - mixture of 40% dimethylacetamide, 30% acetone, and 30% ethanol
Concentration:
0.3, 3.0 and 30% test item in vehicle (DAE 433)
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: dispersion
- Irritation: no
- Lymph node proliferation response: not examined

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:
Stimulation index (SI) is ≥ 3
Response increases in dose-related manner
- Criteria for irritation potential: individual and mean ear weight

TREATMENT PREPARATION AND ADMINISTRATION:
Days 1-3: application of 25 µL test suspension (test item in DAE 433), 1x on day 1, 2, and 3
Days 4 and 5: no treatment
Day 6: Injection of 250 μL of phosphate-buffered saline (PBS) containing 7.5E+5 Bq (ca. 21 µCi) of 3H-methyl thymidine into all test and control mice via the tail vein. Five hours later, the animals were killed.
Positive control substance(s):
other: DNCB (dinitrochlorbenzene, 0.5% (w/v) solution)
Statistics:
Non-parametric Kruskal-Wallis test and then the non-parametric two-group Mann-Whitney rank test (probability level 0.05) were applied to all two-group comparisons [using software Statgraphic ® Centurion (version XV, USA)].
Positive control results:
The positive control substance produced the intended effect. A SI of 18.49 was obtained.
Key result
Parameter:
EC3
Value:
0.585
Key result
Parameter:
SI
Value:
1.54
Test group / Remarks:
0.3% test item in vehicle
Key result
Parameter:
SI
Value:
15.35
Test group / Remarks:
3% test item in vehicle
Key result
Parameter:
SI
Value:
17.06
Test group / Remarks:
30% test item in vehicle
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Measured radioactivity data (DPM) are reported in Table 1 under 'Any other information on results incl. tables'

DETAILS ON STIMULATION INDEX CALCULATION
-Group means were calculated from the radioactivity measurements (DPM) of the individual test animals. Stimulation indices were calculated by dividing the group means of the positive control group and the test groups by the group mean of the negative control group.

EC3 CALCULATION
Equation for EC3 calculation
EC3 = c + [(3 - d) / (b - d] * (a - c),
with a = the lowest concentration in % giving a stimulation index >3;
b = the actual stimulation index caused by a;
c = the highest concentration in % failing to produce a stimulation index of 3;
d = the actual stimulation index caused by c.
Note: The vehicle control data (at SI = 1) should not be used for c and d.

Table 1: Individual and mean radioactivity in cell suspensions (Report Table 4)

Activity (DPM)

Animal No.

NC

(1-5)

PC

(6-10)

30%

(11-15)

3%

(16-20)

0.3%

(21-25)

1

550.98

11374.69

9833.62

7176.23

1061.30

2

485.39

10401.94

9768.19

10503.12

539.22

3

568.93

10347.82

8392.34

6681.56

404.66

4

526.30

8837.38

10204.93

6586.68

1621.48

5

596.74

9493.34

8333.70

10939.31

570.46

 Group mean

545.67

10091.03

9306.56

8377.38

839.42

SI

1.00

18.49

17.06

15.35

1.54

NC = vehicle control; PC = positive control

Table 2: Individual and average ear weights (Report Table 5)

Animal No.

Weight of ear biopsies (milligrams)

NC

(1-6)

PC

(7-12)

30%

(13-18)

3%

(19-24)

0.3%

(25-30)

1

24.5

27.4

29.3

30.3

22.2

2

25.2

27.5

28.8

31.5

23.7

3

23.8

26.5

27.9

28.8

23.6

4

23.4

26.7

28.4

29.2

24.1

5

24.7

27.2

33.6

29.7

23.2

 Group mean

24.32

27.06*

29.60*

29.90*

23.36

SD

0.72

0.44

2.29

1.06

0.72

NC = vehicle control; PC = positive control

* statistically significant with p =< 0.05, Mann-Whitney test

Table 3:  Summary table (Report Table 6)

Group

Radioisotope incorporation
in lymph nodes

Ear weight

Mean DPM

SI

Mean (mg)

NC

545.67

1.0

24.32

PC

10091.03

18.49+

27.06

30%

9306.56

17.06+

29.60

3%

8377.38

15.35+

29.90

0.3%

839.42

1.54

23.36

Bold figures with + = SI values 3

Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
In a LLNA test according to OECD TG 429, test material concentrations of 3% and 30% in vehicle resulted in stimulation indices above 3 (15.35 and 17.06). The EC3 was calculated to 0.585 indicating that the test substance has a high potential for skin sensitisation.
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Principles of method if other than guideline:
Test material is 'Naphtha (petroleum), steam-cracked, C8-10 aromatic hydrocarbon fraction, alkylated and oligomerised' in form of the technical product Novares TL 10.
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
- Name of test material (as cited in study report): Novares TL 10; CAS-no. 71302-83-5 (Hydrocarbons, C9- unsaturated, polymerized)
- Composition of test material, percentage of components: see Section 1.2 Composition
- Lot/batch No.: 28724
- Stability under test conditions: no measured data; based on chemical structure assumed to be stable
- Storage condition of test material: room temperature, exclusion of light
Species:
mouse
Strain:
Balb/c
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Breeding farm VELAZ s.r.o., Koleč u Kladna, Czech Republic
- Age at study initiation: 8 - 10 wks
- Weight at study initiation: 17 - 19 g
- Housing: Macrolon cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 – 70
- Photoperiod (hrs dark / hrs light): 12 / 12

Vehicle:
other: DAE 433 - mixture of 40% dimethylacetamide, 30% acetone, and 30% ethanol
Concentration:
0.3, 3.0 and 30% test item in vehicle (DAE 433)
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: dispersion
- Irritation: no
- Lymph node proliferation response: not examined

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:
Stimulation index (SI) is ≥ 3
Response increases in dose-related manner
- Criteria for irritation potential: individual and mean ear weight

TREATMENT PREPARATION AND ADMINISTRATION:
Days 1-3: application of 25 µL test suspension (test item in DAE 433, 1x on day 1, 2, and 3
Days 4 and 5: no treatment
Day 6: Injection of 250 μL of phosphate-buffered saline (PBS) containing 7.5E+5 Bq (ca. 21 µCi) of 3H-methyl thymidine into all test and control mice via the tail vein. Five hours later, the animals were killed.



Positive control substance(s):
other: DNCB (dinitrochlorbenzene, 0.5% (w/v) solution)
Statistics:
Non-parametric Kruskal-Wallis test and then the non-parametric two-group Mann-Whitney rank test (probability level 0.05) were applied to all two-group comparisons [using software Statgraphic ® Centurion (version XV, USA)].
Positive control results:
The positive control substance produced the intended effect. A SI of 18.49 was obtained.
Key result
Parameter:
EC3
Value:
2.229
Key result
Parameter:
SI
Value:
1.05
Test group / Remarks:
0.3% test item in vehicle
Key result
Parameter:
SI
Value:
3.78
Test group / Remarks:
3% test item in vehicle
Key result
Parameter:
SI
Value:
4.39
Test group / Remarks:
30% test item in vehicle
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Measured radioactivity data (DPM) are reported in Table 1 under 'Any other information on results incl. tables'

DETAILS ON STIMULATION INDEX CALCULATION
-Group means were calculated from the radioactivity measurements (DPM) of the individual test animals. Stimulation indices were calculated by dividing the group means of the positive control group and the test groups by the group mean of the negative control group.

EC3 CALCULATION
Equation for EC3 calculation
EC3 = c + [(3 - d) / (b - d] * (a - c),
with a = the lowest concentration in % giving a stimulation index >3;
b = the actual stimulation index caused by a;
c = the highest concentration in % failing to produce a stimulation index of 3;
d = the actual stimulation index caused by c.
Note: The vehicle control data (at SI = 1) should not be used for c and d.

Table 1: Individual and mean radioactivity in cell suspensions (Report Table 4)

3H-Activity (DPM)

Animal

No.

NC

(1-5)

PC

(6-10)

30%

(11-15)

3%

(16-20)

0.3%

(21-25)

1

550.98

11374.69

1999.50

1476.55

563.51

2

485.39

10401.94

1990.44

2125.60

647.16

3

568.93

10347.82

2943.92

2380.23

576.63

4

526.30

8837.38

2418.17

2337.71

651.87

5

596.74

9493.34

2612.47

1986.59

429.06

 Group

mean

545.67

10091.03

2392.90

2061.34

573.65

SI

1.00

18.49

4.39

3.78

1.05

NC = vehicle control; PC = positive control

Table 2: Individual and average ear weights (Report Table 5)

Weight of ear biopsies (milligrams)

Animal

No.

NC

(1-6)

PC

(7-12)

30%

(13-18)

3%

(19-24)

0.3%

(25-30)

1

24.5

27.4

24.4

23.3

20.1

2

25.2

27.5

24.1

25.7

22.2

3

23.8

26.5

25.3

23.6

20.6

 4

23.4

26.7

25.0

23.7

20.9

5

24.7

27.2

26.9

23.8

23.8

 Group

mean

24.32

27.06*

25.14

24.02

21.52*

SD

0.72

0.44

1.09

0.96

1.49

NC = vehicle control; PC = positive control

* statistically significant with p 0.05, Mann-Whitney test

Table 3:  Summary table (Report Table 6)

Group

Radioisotope incorporation
in lymph nodes

Ear weight

Mean DPM

SI

Mean (mg)

NC

545.67

1.0

24.32

PC

10091.03

18.49+

27.06

30%

2392.90

4.39+

25.14

3%

2061.34

3.78+

24.02

0.3%

573.65

1.05

21.52

Bold figures with + = SI values 3

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
In a LLNA test according to OECD TG 429, test material concentrations of 3.0% and 30% in vehicle resulted in stimulation indices above 3 (3.78 and 4.39). The EC3 was calculated to 2.229 indicating that the test substance has a low to moderate potential for skin sensitisation.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Two technical products of the substance NAF-AO (Novares L 100 and Novares TL 10) were tested for skin sensitisation in GLP compliant mouse local lymph node assays according to OECD TG 429. In both tests, stimulation indices above 3 were determined. EC3 values of 0.585 and 2.229 were calculating for the products Novares L 100 and TL 10, respectively, using the stimulation indices obtained in the studies.

Due to the somewhat different spectrum of constituents, test results of the two product seem to differ to some degree. The study result of the technical product Novares L 100 (lower EC3 value, worst case) is taken as representative of the potential of the substance NAF-AO to produce skin sensitisation.

 

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

There is no experimental data on respiratory sensitisation. But, long-term occupational experience does not provide evidence that the substance may cause respiratory sensitisation.

Justification for classification or non-classification

The substance ‘Naphtha (petroleum), steam-cracked, C8-10 aromatic hydrocarbon fraction, alkylated and oligomerised’ [EC no. 701-299-7] shows a clear potential for skin sensitisation. The lower EC3 value (0.585) of two LLNA tests performed with two technical products of the substance is used for classification (worst-case approach). According to Regulation (EC) 1272/2008, the substance has to be classified as skin sensitising Cat. 1A.