Registration Dossier

Administrative data

Description of key information

Skin sensitisation:

A substance specific skin sensitisation study with naphthenic acids, zinc salts, basic is available. However, the results obtained in the local lymph node assay (LLNA) were found to be equivocal due to observed skin irritation. This is in line with two LLNA assays conducted with naphthenic acid and naphthenic acids, zinc salt, respectively. Guinea pig maximisation tests conducted with naphthenic acids or naphthenic acids, zinc salts showed clear evidence that both substances have skin sensitisation potential. This is supported by a QSAR prediction showing that almost all constituents of naphthenic acid were identified as being skin sensitiser.

Based on a weight of evidence approach and according to the criteria given in Regulation 1272/2008 and its subsequent amendments, naphthenic acids, zinc salts, basic is classified as Skin sensitiser, Cat. 1 H317: May cause an allergic skin reaction. A subclassification into Cat. 1A or 1B is not possible, since the potency of the effects cannot be appropriately assessed with the available information.

Respiratory sensitisation:

No animal respiratory sensitisation study with Naphthenic acids, zinc salts, basic is available. However, respiratory sensitisation data in humans are available and reported in section 7.10.4.

A questionnaire was developed focussing on the potential of Naphthenic acids, zinc salts, basic to cause respiratory (and skin) sensitisation. None of the exposed workers in Europe reported any sign of respiratory sensitisation in the last year and the last decade. Thus, naphthenic acids, zinc salts, basic is not to be classified according to regulation (EC) 1272/2008 and its subsequent amendments for respiratory sensitisation.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Although the information is only available as a published peer reviewed article which does not mention GLP compliance of the studies, enough details are provided to adequately interpret the obtained data. The following information are not given: environmental conditions of the animals, whether females were nulliparous and non-pregnant, acclimatisation period of the animals, body weight at the beginning and the end of the study, historical control data for positive control substance, details on test substances used, animal number per test group is less than 10, details on skin reaction to the induction dose (induction dose should cause mild-to-moderate skin irritation), no second observation has been performed after 72 hours, no details on individual scorings.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Principles of method if other than guideline:
Although no guideline was followed the methods and results are well described allowing a detailed interpretation of the results.
The most important deviation is the number of animals per group. In this test 5 instead of the recommended minimum of 10 animals have been used. Next to this also the non-reporting of the performance of the positive control is deviating from the guideline.
GLP compliance:
not specified
Remarks:
data accessed from publication
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Already existing study. The LLNA 429 was not formally adopted by the OECD until 22 July 2010.
Species:
guinea pig
Strain:
Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Japan SLC (Shizuoka, Japan)
- Age: 6 weeks
Route:
epicutaneous, open
Concentration / amount:
First induction: 1%
Second induction: 25%
No.:
#1
Route:
epicutaneous, open
Vehicle:
other: 0.5% in petroleum ether
Concentration / amount:
0.5% as challenge dose
No. of animals per dose:
5
Details on study design:
6-week-old female Hartley guinea pigs from Japan SLC (Shizuoka, Japan) were used The GPMT was performed as described previously in the literature. 5 animals were used for both sensitization groups. The first induction dose was set at 1%, while the second induction dose was 25%. 2 weeks after the second induction, 0.1 ml aliquots of the test substance in vehicle (0.5% in petroleum ether) was applied to a shaved area of the flank for challenge. 2 days after the challenge, each site was scored according to the criteria of Sato et al.

Additional information:
As mentioned in the material and method section, the test was performed as described in Nakamura et al. (1994) (see linked reference). In this reference the following additional information is given:
- Exposure duration: 24 hours
- challenge was perfomed by the open patch test method
- the selected challenge concentration was lower than the irritant concentrations
- time until challenge: 21 days after the initial induction dose (this leads to the conclusion that 7 days were between the first and second induction dose)
- the total erythema and edema scores were summed in each group of animals. The total score was divided by the number of animals in the group to give the mean response induced by the given concentration for induction and challenge. The number of positive sites was divided by the numer of animals of the group to give the sensitization rate.

Reference:
* Sato Y. Katsumura Y. Ichikawa H. Kobayashi T. Kozuka T. Morikawa F. Ohta S. A modilied tcchnique of guinea pig testing to identify delayed hypersensitivity allergens. Contact Dermatitis 1981: 7: 225 237.
Challenge controls:
none
Positive control substance(s):
not specified
Positive control results:
Not reported
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Vehicle
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
0.5%
No. with + reactions:
3
Total no. in group:
5
Clinical observations:
median skin reaction score: 1
Remarks on result:
other: Naphthenic acid, zinc salts
Reading:
1st reading
Hours after challenge:
48
Group:
positive control
Remarks on result:
not measured/tested
Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
A GPMT test in 6-week-old female Hartley guinea pigs was performed with Naphthenic acid, zinc salts. The first induction dose was set at 1%, while the second induction dose was 25%. Two weeks after the second induction, 0.1 ml aliquots of the test substances in vehicle (0.5% in petroleum ether) was applied to a shaved area of the flank for challenge. Each site was scored 48 hours after challenge. Naphthenic acid, zinc salts resulted in 3/5 animals with clear reactions at 48 hours, therefore the substance is considered to be sensitizing for skin.

Although the information is only available as a published peer reviewed article which does not mention GLP compliance of the studies and some information were not provided, enough details are provided to adequately interpret the obtained data and to conclude that both substances seem to have skin sensitising potential.

The following information are not given: environmental conditions of the animals, whether females were nulliparous and non-pregnant, acclimatisation period of the animals, body weight at the beginning and the end of the study, historical control data for positive control substances, details on test substance used, animal number per test group is less than 10, details on skin reaction to the induction dose (induction dose should cause mild-to-moderate skin irritation), no second observation has been performed after 72 hours, no details on individual scorings.
Executive summary:

A GPMT test in 6-week-old female Hartley guinea pigs was performed with Naphthenic acid, zinc salts. The first induction dose was set at 1%, while the second induction dose was 25%. Two weeks after the second induction, 0.1 ml aliquots of the test substance in vehicle (0.5% in petroleum ether) was applied to a shaved area of the flank for challenge. Each site was scored 48 hours after challenge. Naphthenic acid, zinc salts resulted in 3/5 animals with clear reactions at 48 hours, therefore the substance is considered to be sensitizing for skin. However, the number of animals per group is too low to obtain information about the potency of the sensitisation effect.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Although the information is only available as a published peer reviewed article which does not mention GLP compliance of the studies, enough details are provided to adequately interprete the obtained data.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Principles of method if other than guideline:
In the materials and methods section reference is made to the current OECD 429 guideline method. Nevertheless the applied method is the non radioactive BrdU read-out which is similar to the current OECD 442B guideline. The general study set-up, number of animals per group and exposure has been described as conform the current guideline.
The most inmportant deviations are the use of BALB-C in stead of CBA/Ca or CBA/J mouse strain and the non-reporting of the performance of a negative and positive control.
GLP compliance:
not specified
Remarks:
data accessed from publication
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
Balb/c
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: CLEA Japan (Tokyo, Japan)
- Age: 6-8 weeks
Vehicle:
other: petroleum ether and olive oil 4:1
Concentration:
25 µL containing
Naphthenic acid: 3, 10, 30 and 50%
No. of animals per dose:
4
Details on study design:
6- to 8-week-old female BALB/c mice from CLEA Japan (Tokyo, Japan) were used. The assay was performed by the modified method of the standard LLNA reported by Takeyoshi et al. with some modificatios. The modified method has been reported to have almost comparable sensitivity to the radioactive original method using dinitrochlorobenzene as a standard sensitizer.
In brief, groups of mice (n = 4) were exposed to 25 μL of various concentrations of the test substance in a vehicle (petroleum ether and olive oil, 4 : 1 ) through application to the dorsum of both ears for 3 consecutive days (days 0-2). On day 4, 5- bromo-7-deoxyuridine (BrdU) was administered intraperitoneally to each mouse. The next day, a pair of auricular lymph nodes from each mouse was excised. After counting the total cell numbers from each mouse, BrdU concentrations were measured by enzyme-linked immunosorbent assay. Total lymph node cell count in a dosed animal divided by mean lymph node cell count in a control group was designated as a cellularity index, whereas BrdU incorporation per unit number of cells from a dosed animal divided by mean BrdU incorporation from a control group was used as a BrdU-incorporation index. A LLNA stimulation index was calculated by multiplying the cellularity index by the BrdU-incorporation index. At the time of lymph node excision, the thickness of the ears was measured with a digital micrometer (Mitutoyo Corporation, Kawasaki, Japan) at the edge of the right pinna. Ear thickness of a dosed animal divided by mean ear thickness of a control group was designated as an ear-thickness index.
Positive control substance(s):
not specified
Statistics:
In the LLNA, index values for each dosed group and vehicle-control group were compared via Dunnett’s or Steel’s multiple comparison method using a STATLIGHT software package (Yukms, Tokyo, Japan).
Positive control results:
Not reported
Key result
Parameter:
SI
Value:
3
Variability:
estimated value based on the graphic shown in the publication
Test group / Remarks:
50 % Naphthenic acids
Remarks on result:
other: Although a significant increase of the stimulation index was observed at the highest dose, this was also accompanied with an increase in the irritation index, by which the test results of the LLNA for naphthenic acid are not conclusive.
Parameter:
SI
Value:
1.5
Variability:
estimated value based on the graphic shown in the publication
Test group / Remarks:
30% Naphthenic acids
Remarks on result:
other: Although a significant increase of the stimulation index was observed at this dose, this was also accompanied with an increase in the irritation index, by which the test results of the LLNA for naphthenic acid are not conclusive.
Parameter:
SI
Value:
1
Variability:
estimated value based on the graphic shown in the publication
Test group / Remarks:
10% Naphthenic acids
Parameter:
SI
Value:
1.2
Variability:
estimated value based on the graphic shown in the publication
Test group / Remarks:
3% Naphthenic acids
Interpretation of results:
other: not conclusive
Conclusions:
Due to the increase of the stimulation index which was accompanied with an increase in the irritation index, the results are not conclusive about the sensitizing properties of naphthenic acids under the conditions of the LLNA test.
Executive summary:

In an LLNA test, 6- to 8-week-old female BALB/c mice (n=4/group) were exposed to 25 μL of various concentrations of Naphthenic acids in petroleum ether and olive oil (4 : 1) through application to the dorsum of both ears for 3 consecutive days. On day 4, 5- bromo-7-deoxyuridine (BrdU) was administered intraperitoneally to each mousse for counting the total cell numbers from each mouse. BrdU concentrations were measured by enzyme-linked immunosorbent assay. Total lymph node cell count in a dosed animal divided by mean lymph node cell count in a control group was designated as a cellularity index, whereas BrdU incorporation per unit number of cells from a dosed animal divided by mean BrdU incorporation from a control group was used as a BrdU-incorporation index. A LLNA stimulation index was calculated by multiplying the cellularity index by the BrdU-incorporation index. At the time of lymph node excision, the thickness of the ears was measured with a digital micrometer at the edge of the right pinna. Ear thickness of a dosed animal divided by mean ear thickness of a control group was designated as an ear-thickness index. There was an increase of the stimulation index for both substances, which was accompanied with an increase in the irritation index, therefore the results are not conclusive about the sensitizing properties of naphthenic acid under the conditions of the LLNA test.

Endpoint:
skin sensitisation: in chemico
Remarks:
(Q)SAR
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Skin sensitisation properties have been estimated using the validated VEGA modelling tool (Istituto di Ricerche Farmacologiche Mario Negri).
Justification for type of information:
[1]VEGA skin sensitisation classification model version 2.1.1. This is an
online beta version. The VEGA application is a Java web application that
allows the access to all the toxicity predictive models developed within the
VEGA project. http://www.vega-qsar.eu/use-qsar.html
[2]VegaNic Skin sensitisation classification model version 2.1.1. download
This is the downloadable version of the VEGA mutagenicity classification
model version 2.1.7. It is only downloadable after registration.
http://www.vegaqsar. eu/download.html
Guideline:
other: Guidance on information requirements and chemical safety assessment Chapter R.7a: Endpoint specific guidance, non-testing data for skin sensitisation, p 264-264
Principles of method if other than guideline:
Skin sensitisation was predicted using the VEGA skin sensitisation model which is an extension of the original CAESAR model.
GLP compliance:
no
Remarks:
not applicable for QSARs
Details on the study design:
Skin sensitisation (In chemico test system) - Details on study design:
Based on the analysis results (IUCLID section 1.4) of representative naphthenic acids samples, a list of potential chemical components was compiled. The criteria for choosing these specific structures were the occurrence of C-numbers, the number of rings and their relative share in the analysed samples. The biodegradation of these structures is estimated in Biowin (v4.10).
Remarks on result:
other: qualitative QSAR calculations

Since naphthenic acids can adopt a wide variety of structures, QSARs are peformed for a large number of different structures. The structures are chosen in order to represent the complete composition of the mixture of naphthenic acids. From the QSARs results it can be demonstrated that naphthenic acids are skin sensitisers.

In following table all results from the performed QSARs are given. The prediction along with the applicability domain index (ADI) is given.

ADI>=0.8: in applicability domain

0.8>ADI>=0.6: could be out of applicability domain

ADI <0.6: out of applicability domain

C-number Ring Branch SMILES Mw prediction ADI
C6 - - C(=O)(O)CCCCC 116,16 sensitiser 0,738
C7 - - C(=O)(O)CCCCCC 130,19 sensitiser 0,724
C8 - - C(=O)(O)CCCCCCC 144,22 sensitiser 0,853
C8 1 pentane - C(=O)(O)CCC1CCCC1 142,2 sensitiser 0,844
C9 - methyl C(=O)(O)CCCC(C)CCC 158,24 sensitiser 0,853
C10 - ethyl C(=O)(O)CCCC(CCC)CC 172,27 sensitiser 0,85
C10 1 pentane - C(=O)(O)CCC1C(CC)CCC1 170,25 sensitiser 0,847
C11 1 pentane - C(=O)(O)CCC1C(CCC)CCC1 184,28 sensitiser 0,847
C12 1 hexane - C(=O)(O)CCC1C(CCC)CCCC1 198,31 sensitiser 0,847
C12 1 pentane - C(=O)(O)CCC1C(CCCC)CCC1 198,31 sensitiser 0,847
C12 2 pentanes fused - C(=O)(O)CCC1C2C(C)CCC2CC1 196,29 sensitiser 0,858
C13 2 pentanes - C(=O)(O)CCC1C(C2CCCC2)CCC1 210,32 sensitiser 0,853
C13 pentane hexane fused   O=C(O)CCC2CCCC1CCC(C)C12 210,32 sensitiser 0,86
C14 1 hexane - O=C(O)C1C(CCCCCCCC)CCC1 226,36 sensitiser 0,845
C14 2 pentanes - C(=O)(O)CCC1CC(C2C(C)CCC2)CC1 224,35 sensitiser 0,852
C14 2 pentanes fused - C(=O)(O)CCCC1C2C(CC)CCC2CC1 224,35 sensitiser 0,86
C15 - propyl C(=O)(O)CCCCC(CCCCCC)CCC 242,41 sensitiser 0,857
C15 1 pentane ethyl C(=O)(O)CCC1C(CC(CCC)CC)CCC1 240,39 sensitiser 0,844
C15 2 pentanes - C(=O)(O)CCC1C(CC2CC(C)CC2)CCC1 238,37 sensitiser 0,852
C15 2 hexanes fused   O=C(O)CCC1CCC2CCCC(CC)C2C1 238,37 sensitiser 0,86
C15 3 pentanes fused - C(=O)(O)CCCC1C2C3C(CC2CC1)CCC3C 250,38 non-sensitiser 0
C16 1 pentane - C(=O)(O)CCC1C(CCCCCCCC)CCC1 254,42 sensitiser 0,844
C16 2 pentanes - C(=O)(O)CCC1CC(C2C(CCC)CCC2)CC1 252,4 sensitiser 0,852
C16 2 hexane - C(=O)(O)C1C(CCC2C(C)CCCC2)CCCC1 252,4 sensitiser 0,852
C16 3 pentanes of which 2 fused - C(=O)(O)CC1C2C(CC3CCCC3)CCC2CC1 250,38 sensitiser 0,868
C17 1 pentane - C(=O)(O)CCC1C(CCCCCCCCC)CCC1 268,44 sensitiser 0,843
C17 4 pentanes fused - C12(C3C(CC(=O)O)CCC3CC1)C1C(CCC1)CC2 262,4 sensitiser 0,859
C18 1 hexane propyl C(=O)(O)CCC1C(CC(CCCC)CCC)CCCC1 282,47 sensitiser 0,842
C18 3 pentanes of which 2 fused - C(=O)(O)CCCCC1C2C(C3CCCC3)CCC2CC1 278,44 sensitiser 0,866
C19 2 pentanes fused ethyl C(=O)(O)CCCCC1C2C(C(CCC)CC)CCC2CC1 294,48 sensitiser 0,856
C19 2 pentane propyl C(=O)(O)CC1CC(CC(CCC2CCCC2)CCC)CC1 294,48 sensitiser 0,849
C25 2 hexane propyl C(=O)(O)CCCC(CCC1C(CCC2C(C)CCCC2)CCCC1)CCC 378,64 sensitiser 0,837
C30 2 hexane propyl C(=O)(O)CCC(CCCC1C(CCC2C(CCCCCC)CCCC2)CCCC1)CCC 448,78 sensitiser 0
C30 3 hexane - C(=O)(O)CCCCCCC1C(CCC2C(CCCC3CCCCC3)CCCC2)CCCC1 446,76 sensitiser 0
C30 3 hexanes fused ethyl-ethyl O=C(O)CCCCC(CC)CCC1CC2CCCC3CC(CCC(CC)CC)CC(C1)C23 446,76 non-sensitiser 0
C30 2 hexanes fused 1 not propyl O=C(O)CCCC(CCC)CCCC1CCC2CC(CCC2C1)CC3CCCC(C)C3 432,74 sensitiser 0

As can be seen from the results in the table above, most of the molecules are indicated to be a sensitiser. Only two molecules are indicated to be a non-sensitiser with a low ADI. Most molecules fall in the applicability domain (ADI >=0.8) and thus the prediction can be assumed to be reliable.

Validity of the model

1.Defined endpoint: Skin sensitisation

2. Unambiguous algorithm: Adaptive fuzzy partition models have been used to generate models.

The AFP method allocates degrees of membership of the different classes for each compound within a 0 to 1 range. Then, a compound is attributed to a given class if its degree of membership is greater than 0.5. The percentage

of compounds correctly predicted is computed by comparing their experimental and predicted classes.

3. Applicability domain: The applicability domain is indicated by a globa applicability domain index (ADI) which takes into account similar molecules with known experimental values, accuracy of prediction for similar molecules, concordance for similar molceuls, atom centered fragments similary check, model descriptors range chek. The ADI has values from 0 (worst case) to 1 (best case)
4. Statistical characteristics: training set:

n= 167, accuracy = 0.92; Specificity = 0.82; Sensitivity = 0.95

test set :

n = 42; Accuracy = 0.90; Specificity = 0.75; Sensitivity = 0.94

5. Mechanistic interpretation:
Adequacy of prediction:
Most of the naphtenic acids fall within the applicability domain described above. 29 out of the 36 molecules fall within the applicability domain. For these 29 molecules VEGA performs a reliable prediction for skin sensitisation.

Conclusions:
According to the VEGA model for skin sensitisation, naphthenic acids are sensitising.
Executive summary:

Skin sensitisation was predicted using the VEGA sensitisation model which is an extension of the original CAESAR model. Since naphthenic acids do not have a fixed composition, the prediction was performed on a broad selection of different possible structures in accordance with the substance identification. As can be seen from the results, most of the molecules were indicated to be a sensitiser. Only two molecules were indicated to be a non-sensitiser with a low applicability domain index (ADI). Most molecules fell in the applicability domain (with ADI >=0.8) and thus the prediction can be assumed to be reliable.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
The relative humidity in the animal room was between approximately a minimum of 27 - 64% instead of 45 – 65% for several hours on five not subsequent days. This deviation to the study plan, however, does not affect the validity of the study.
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
The animals were distributed into the test groups at random. All animals belonging to the same experimental group were kept in one cage. In the main experiment, the animals were identified by tail tags. In the pre-experiment, animals were identified by cage number. At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.
The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.
Housing: group
Cage Type: Makrolon Type II (pre-test) / III (main study), with wire mesh top
Bedding: granulated soft wood bedding
Feed: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
Water: tap water, ad libitum
Environment: temperature 22 + 2°C relative humidity approx. 45-65% (except for few hours on five not subsequent days, see deviation section) artificial light 6.00 a.m. - 6.00 p.m.
Vehicle:
methyl ethyl ketone
Concentration:
0, 0.5, 1 and 2.5%
No. of animals per dose:
4
Positive control substance(s):
other: no positive control
Statistics:
The mean values and standard deviations were calculated in the body weight tables. However, both biological and statistical significance were considered together.
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
0% (control group)
Key result
Parameter:
SI
Value:
1.36
Test group / Remarks:
0.5%
Key result
Parameter:
SI
Value:
4.72
Test group / Remarks:
1%
Key result
Parameter:
SI
Value:
2.43
Test group / Remarks:
2.5%
Key result
Parameter:
EC3
Remarks on result:
not determinable
Remarks:
The EC3 value could not be calculated, since the S.I. of the mid dose is above the threshold value of 3 and above the value of the high dose.
Cellular proliferation data / Observations:
Viability / Mortality
No deaths occurred during the study period.

Clinical Signs
No signs of systemic toxicity were observed during the study period. On day 4 to 6, the animals treated with a test item concentration of 1 and 2.5% showed an erythema of the ear skin (Score 1). Animals treated with 0.5% test item concentration did not show any signs of local skin irritation.

Body Weights
The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age. The individual body weight values are included in Annex 2.

In order to study a possible skin sensitising potential of VP 112/18, three groups each of four female mice were treated once daily with the test item at concentrations of 0.5, 1 and 2.5% (w/w) in MEK by topical application to the dorsum of each ear for three consecutive days. The highest concentration tested was the highest concentration that could be achieved whilst avoiding excessive local skin irritation as confirmed by three pre-experiments. A control group of four mice was treated with the vehicle (MEK) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a β-scintillation counter.

All treated animals survived the scheduled study period and no signs of systemic toxicity were observed. On days 4 to 6, the animals treated with a test item concentration of 1 and 2.5% showed an erythema of the ear skin (Score 1). Animals treated with 0.5% test item concentration did not show any signs of local skin irritation. A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in a 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value. In this study Stimulation Indices (S.I.) of 1.36, 4.72 and 2.43 were determined with the test item at concentrations of 0.5, 1 and 2.5% in MEK, respectively. A conventional dose response showing increased sensitizing effects with increasing VP 112/18 concentrations was not observed. The Stimulation Index obtained in the mid dose group was above the threshold index of 3. However, the Stimulation Index obtained for the high dose group was below that of the mid dose group and below the threshold index of 3. An EC3 value could therefore not be calculated. Based on the observed irritation, it cannot be ruled out (although signs of irritation were not excessive) that the irritant potential of the test item might have contributed to the increase in S.I. values observed in the mid and high dose group.

Interpretation of results:
other: not conclusive
Conclusions:
The results obtained with the test item VP 112/18 were found to be equivocal under the test conditions of this study.
Executive summary:

In the study the test item VP 112/18 formulated in MEK was assessed for its possible skin sensitising potential. For this purpose a local lymph node assay was performed using test item concentrations of 0.5, 1 and 2.5%. The highest concentration tested was the highest concentration that could be achieved whilst avoiding excessive local skin irritation as confirmed by three pre-experiments. The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. On days 4 to 6, the animals treated with a test item concentration of 1 and 2.5% showed an erythema of the ear skin (Score 1). Animals treated with 0.5% test item concentration did not show any signs of local skin irritation. In this study Stimulation Indices (S.I.) of 1.36, 4.72 and 2.43 were determined with the test item at concentrations of 0.5, 1 and 2.5% in MEK, respectively. A conventional dose response showing increased sensitizing effects with increasing VP 112/18 concentrations was not observed. The Stimulation Index obtained in the mid dose group was above the threshold index of 3. However, the Stimulation Index obtained for the high dose group was below that of the mid dose group and below the threshold index of 3. An EC3 value could therefore not be calculated. Based on the observed irritation, it cannot be ruled out (although signs of irritation were not excessive) that the irritant potential of the test item might have contributed to the increase in S.I. values observed in the mid and high dose group. Therefore, the results obtained with the test item VP 112/18 were found to be equivocal.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Guideline compliant study, used in EU risk assessment for zinc oxide 2004
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Already existing study. The LLNA 429 was not formally adopted by the OECD until 22 July 2010.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
see reference
Route:
intradermal
Vehicle:
water
Concentration / amount:
see details on study design
Route:
other: epidermal
Vehicle:
water
Concentration / amount:
see details on study design
No. of animals per dose:
10 in each test in main study
5 controls in each test
Details on study design:
In a third well-performed maximisation test, conducted according to the same guidelines and with the same experimental design, another analytical grade zinc oxide was tested (Zincweiß Pharma A; purity 99.9%). The only difference with the studies described for purity ZnO 99.69% was the intradermal induction concentration, which was 2% as for Zincweiß Pharma A this was considered the highest concentration that could reproducibly be injected. In this test no skin reactions were evident in both experimental and control animals, hence a 0% sensitisation rate for Zincweiß Pharma A. White staining of the treated skin by the test substance was observed in some animals 24 and 48 hours after challenge
Challenge controls:
see details on study designs
Positive control substance(s):
yes
Key result
Reading:
1st reading
Group:
test chemical
Dose level:
2 %
No. with + reactions:
0
Total no. in group:
10
Key result
Group:
positive control
Remarks on result:
other: results not specified
Key result
Reading:
1st reading
Group:
negative control
Dose level:
2 %
No. with + reactions:
0
Total no. in group:
5
Interpretation of results:
GHS criteria not met
Conclusions:
Not sensitising
Executive summary:

In a third well-performed maximisation test, conducted according to the same guidelines and with the same experimental design, another analytical grade zinc oxide was tested (Zincweiß Pharma A; purity 99.9%). The only difference with the previous studies described for zinc oxide of purity 99.69% was the intradermal induction concentration, which was 2% as for Zincweiß Pharma A this was considered the highest concentration that could reproducibly be injected. In this test no skin reactions were evident in both experimental and control animals, hence a 0% sensitisation rate for Zincweiß Pharma A. White staining of the treated skin by the test substance was observed in some animals 24 and 48 hours after challenge.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Guideline compliant study, used in EU risk assessment for zinc oxide 2004
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Already existing study. The LLNA 429 was not formally adopted by the OECD until 22 July 2010.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
see reference
Route:
intradermal
Vehicle:
water
Concentration / amount:
see details on study design
Route:
other: epidermal
Vehicle:
water
Concentration / amount:
see details on study design
No. of animals per dose:
10 in each test in main study
5 controls in each test
Details on study design:
Based on the results of a preliminary study, in the main studies experimental animals (10 in each test) were intradermally injected with a 20% concentration and epidermally exposed to a 50% concentration (i.e. the highest practically feasible concentration). Control animals (5 in each test) were similarly treated, but with vehicle (water) alone. Approximately 24 hours before the epidermal induction exposure all animals were treated with 10% SDS. Two weeks after the epidermal application all animals were challenged with a 50% test substance concentration and the vehicle.
Challenge controls:
see details on study designs
Positive control substance(s):
yes
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
50 %
No. with + reactions:
4
Total no. in group:
10
Clinical observations:
Study 1
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50 %. No with. + reactions: 4.0. Total no. in groups: 10.0. Clinical observations: Study 1.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
50 %
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
Study 1
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 50 %. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: Study 1.
Key result
Reading:
1st reading
Group:
test chemical
Dose level:
50 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Study 2
Remarks on result:
other: Reading: 1st reading. Group: test group. Dose level: 50 %. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Study 2.
Key result
Reading:
1st reading
Group:
negative control
Dose level:
50 %
No. with + reactions:
1
Total no. in group:
5
Clinical observations:
Study 2
Remarks on result:
other: Reading: 1st reading. Group: negative control. Dose level: 50 %. No with. + reactions: 1.0. Total no. in groups: 5.0. Clinical observations: Study 2.
Key result
Group:
positive control
Remarks on result:
other: results not specified
Conclusions:
conflicting results
Executive summary:

The skin sensitising potential of zinc oxide (purity 99.69%) was investigated in female Dunkin Hartley guinea pigs in two well-performed maximisation tests, conducted according to Directive 96/54/EC B.6 and OECD guideline 406. Based on the results of a preliminary study, in the main studies experimental animals (10 in each test) were intradermally injected with a 20% concentration and epidermally exposed to a 50% concentration (i.e. the highest practically feasible concentration). Control animals (5 in each test) were similarly treated, but with vehicle (water) alone. Approximately 24 hours before the epidermal induction exposure all animals were treated with 10% SDS. Two weeks after the epidermal application all animals were challenged with a 50% test substance concentration and the vehicle.In the first study, in response to the 50% test substance concentration skin reactions of grade 1 were observed in 4/10 experimental animals 24 hours after the challenge (40% sensitisation rate), while no skin reactions were evident in the controls. In contrast, in the second study no skin reactions were evident in the experimental animals (0% sensitisation rate), while a skin reaction grade 1 was seen in one control animal. The skin reaction observed in one control animal is most likely sign of non-specific irritation.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Although the information is only available as a published peer reviewed article which does not mention GLP compliance of the studies, enough details are provided to adequately interpret the obtained data. The following information are not given: environmental conditions of the animals, whether females were nulliparous and non-pregnant, acclimatisation period of the animals, body weight at the beginning and the end of the study, historical control data for positive control substances, details on test substance used, animal number per test group is less than 10, details on skin reaction to the induction dose (induction dose should cause mild-to-moderate skin irritation), no second observation has been performed after 72 hours, no details on individual scorings.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Principles of method if other than guideline:
Although no guideline was followed the methods and results are well described allowing a detailed interpretation of the results.
The most important deviation is the number of animals per group. In this test 5 instead of the recommended minimum of 10 animals have been used. Next to this also the non-reporting of the performance of the positive control is deviating from the guideline.
GLP compliance:
not specified
Remarks:
data accessed from publication
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Already existing study. The LLNA 429 was not formally adopted by the OECD until 22 July 2010.
Species:
guinea pig
Strain:
Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Japan SLC (Shizuoka, Japan)
- Age: 6 weeks
Route:
epicutaneous, open
Concentration / amount:
First induction: 1%
Second induction: 25%
No.:
#1
Route:
epicutaneous, open
Vehicle:
other: 0.5% in petroleum ether
Concentration / amount:
0.5% as challenge dose
No. of animals per dose:
5
Details on study design:
6-week-old female Hartley guinea pigs from Japan SLC (Shizuoka, Japan) were used The GPMT was performed as described previously in the literature. 5 animals were used for both sensitization groups. The first induction dose was set at 1%, while the second induction dose was 25%. 2 weeks after the second induction, 0.1 ml aliquots of the test substance in vehicle (0.5% in petroleum ether) was applied to a shaved area of the flank for challenge. 2 days after the challenge, each site was scored according to the criteria of Sato et al.

Additional information:
As mentioned in the material and method section, the test was performed as described in Nakamura et al. (1994) (see linked reference). In this reference the following additional information is given:
- Exposure duration: 24 hours
- challenge was perfomed by the open patch test method
- the selected challenge concentration was lower than the irritant concentrations
- time until challenge: 21 days after the initial induction dose (this leads to the conclusion that 7 days were between the first and second induction dose)
- the total erythema and edema scores were summed in each group of animals. The total score was divided by the number of animals in the group to give the mean response induced by the given concentration for induction and challenge. The number of positive sites was divided by the numer of animals of the group to give the sensitization rate.

Reference:
* Sato Y. Katsumura Y. Ichikawa H. Kobayashi T. Kozuka T. Morikawa F. Ohta S. A modilied tcchnique of guinea pig testing to identify delayed hypersensitivity allergens. Contact Dermatitis 1981: 7: 225 237.
Challenge controls:
none
Positive control substance(s):
not specified
Positive control results:
Not reported
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Vehicle
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
0.5%
No. with + reactions:
4
Total no. in group:
5
Clinical observations:
median skin reaction score: 1
Remarks on result:
other: Naphthenic acid
Reading:
1st reading
Hours after challenge:
48
Group:
positive control
Remarks on result:
not measured/tested
Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
A GPMT test in 6-week-old female Hartley guinea pigs was performed with Naphthenic acids. The first induction dose was set at 1%, while the second induction dose was 25%. Two weeks after the second induction, 0.1 ml aliquots of the test substances in vehicle (0.5% in petroleum ether) was applied to a shaved area of the flank for challenge. Each site was scored 48 hours after challenge. Naphthenic acid resulted in 4/5 animals with clear reactions at 48 hours, therefore the substance is considered to be sensitizing for skin.

Although the information is only available as a published peer reviewed article which does not mention GLP compliance of the studies and some information were not provided, enough details are provided to adequately interpret the obtained data and to conclude that both substances seem to have skin sensitising potential.

The following information are not given: environmental conditions of the animals, whether females were nulliparous and non-pregnant, acclimatisation period of the animals, body weight at the beginning and the end of the study, historical control data for positive control substances, details on test substance used, animal number per test group is less than 10, details on skin reaction to the induction dose (induction dose should cause mild-to-moderate skin irritation), no second observation has been performed after 72 hours, no details on individual scorings.
Executive summary:

A GPMT test in 6-week-old female Hartley guinea pigs was performed with Naphthenic acids. The first induction dose was set at 1%, while the second induction dose was 25%. Two weeks after the second induction, 0.1 ml aliquots of the test substances in vehicle (0.5% in petroleum ether) was applied to a shaved area of the flank for challenge. Each site was scored 48 hours after challenge. Naphthenic acid resulted in 4/5 animals with clear reactions at 48 hours, therefore the substances is considered to be sensitizing for skin. However, the number of animals per group is too low to obtain information about the potency of the sensitisation effect.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Although the information is only available as a published peer reviewed article which does not mention GLP compliance of the studies, enough details are provided to adequately interprete the obtained data.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Principles of method if other than guideline:
In the materials and methods section reference is made to the current OECD 429 guideline method. Nevertheless the applied method is the non radioactive BrdU read-out which is similar to the current OECD 442B guideline. The general study set-up, number of animals per group and exposure has been described as conform the current guideline.
The most inmportant deviations are the use of BALB-C in stead of CBA/Ca or CBA/J mouse strain and the non-reporting of the performance of a negative and positive control.
GLP compliance:
not specified
Remarks:
data accessed from publication
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
Balb/c
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: CLEA Japan (Tokyo, Japan)
- Age: 6-8 weeks
Vehicle:
other: petroleum ether and olive oil 4:1
Concentration:
25 µL containing
Naphthenic acid, zinc salt: 0.3, 1, 3 and 10%
No. of animals per dose:
4
Details on study design:
6- to 8-week-old female BALB/c mice from CLEA Japan (Tokyo, Japan) were used. The assay was performed by the modified method of the standard LLNA reported by Takeyoshi et al. with some modificatios. The modified method has been reported to have almost comparable sensitivity to the radioactive original method using dinitrochlorobenzene as a standard sensitizer.
In brief, groups of mice (n = 4) were exposed to 25 μL of various concentrations of the test substance in a vehicle (petroleum ether and olive oil, 4 : 1 ) through application to the dorsum of both ears for 3 consecutive days (days 0-2). On day 4, 5- bromo-7-deoxyuridine (BrdU) was administered intraperitoneally to each mouse. The next day, a pair of auricular lymph nodes from each mouse was excised. After counting the total cell numbers from each mouse, BrdU concentrations were measured by enzyme-linked immunosorbent assay. Total lymph node cell count in a dosed animal divided by mean lymph node cell count in a control group was designated as a cellularity index, whereas BrdU incorporation per unit number of cells from a dosed animal divided by mean BrdU incorporation from a control group was used as a BrdU-incorporation index. A LLNA stimulation index was calculated by multiplying the cellularity index by the BrdU-incorporation index. At the time of lymph node excision, the thickness of the ears was measured with a digital micrometer (Mitutoyo Corporation, Kawasaki, Japan) at the edge of the right pinna. Ear thickness of a dosed animal divided by mean ear thickness of a control group was designated as an ear-thickness index.
Positive control substance(s):
not specified
Statistics:
In the LLNA, index values for each dosed group and vehicle-control group were compared via Dunnett’s or Steel’s multiple comparison method using a STATLIGHT software package (Yukms, Tokyo, Japan).
Positive control results:
Not reported
Key result
Parameter:
SI
Value:
3.5
Variability:
estimated value based on the graphic shown in the publication
Test group / Remarks:
10 % Naphthenic acid, zinc salt
Remarks on result:
other: See remarks
Remarks:
Although a significant increase of the stimulation index was observed, this was also accompanied with an increase in the irritation index, by which the test results of the LLNA for naphthenic acid, zinc salt are not conclusive.
Parameter:
SI
Value:
4.1
Variability:
estimated value based on the graphic shown in the publication
Test group / Remarks:
3 % Naphthenic acid, zinc salt
Remarks on result:
other: see remarks
Remarks:
Although a significant increase of the stimulation index was observed, this was also accompanied with an slight increase in the irritation index, by which the test results of the LLNA for naphthenic acid, zinc salt are not conclusive.
Parameter:
SI
Value:
1.2
Variability:
estimated value based on the graphic shown in the publication
Test group / Remarks:
1% Naphthenic acids, zinc salts
Parameter:
SI
Value:
1.05
Variability:
estimated value based on the graphic shown in the publication
Test group / Remarks:
0.3% Naphthenic acids, zinc salts
Interpretation of results:
other: not conclusive
Conclusions:
Due to the increase of the stimulation index which was accompanied with an increase in the irritation index, the results are not conclusive about the sensitizing properties of naphthenic acid, zinc salts under the conditions of the LLNA test.
Executive summary:

In an LLNA test, 6- to 8-week-old female BALB/c mice (n=4/group) were exposed to 25 μL of various concentrations of Naphthenic acids, zinc salts in petroleum ether and olive oil (4 : 1) through application to the dorsum of both ears for 3 consecutive days. On day 4, 5- bromo-7-deoxyuridine (BrdU) was administered intraperitoneally to each mousse for counting the total cell numbers from each mouse. BrdU concentrations were measured by enzyme-linked immunosorbent assay. Total lymph node cell count in a dosed animal divided by mean lymph node cell count in a control group was designated as a cellularity index, whereas BrdU incorporation per unit number of cells from a dosed animal divided by mean BrdU incorporation from a control group was used as a BrdU-incorporation index. A LLNA stimulation index was calculated by multiplying the cellularity index by the BrdU-incorporation index. At the time of lymph node excision, the thickness of the ears was measured with a digital micrometer at the edge of the right pinna. Ear thickness of a dosed animal divided by mean ear thickness of a control group was designated as an ear-thickness index. There was an increase of the stimulation index, which was accompanied with an increase in the irritation index, therefore the results are not conclusive about the sensitizing properties of naphthenic acid, zinc salts under the conditions of the LLNA test.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Skin sensitisation:

A substance specific skin sensitisation study with naphthenic acids, zinc salts, basic, is available (Dony, 2015). This test was performed using test item concentrations of 0.5, 1 and 2.5%. The highest concentration tested was the highest concentration that could be achieved whilst avoiding excessive local skin irritation as confirmed by three pre-experiments. The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. On days 4 to 6, the animals treated with a test item concentration of 1 and 2.5% showed an erythema of the ear skin (Score 1). Animals treated with 0.5% test item concentration did not show any signs of local skin irritation. In this study Stimulation Indices (S.I.) of 1.36, 4.72 and 2.43 were determined with the test item at concentrations of 0.5, 1 and 2.5% in MEK, respectively. A conventional dose response showing increased sensitizing effects with increasing VP 112/18 concentrations was not observed. The Stimulation Index obtained in the mid dose group was above the threshold index of 3. However, the Stimulation Index obtained for the high dose group was below that of the mid dose group and below the threshold index of 3. An EC3 value could therefore not be calculated. Based on the observed irritation, it cannot be ruled out (although signs of irritation were not excessive) that the irritant potential of the test item might have contributed to the increase in S.I. values observed in the mid and high dose group. Therefore, the results obtained with the test item VP 112/18 were found to be equivocal.

 

These results are in line with the two local lymph node assays available with naphthenic acid, zinc salts and naphthenic acids (Yamano, 2006). 6- to 8-week-old female BALB/c mice (n=4/group) were exposed to 25 μL of various concentrations of Naphthenic acids, zinc salts or naphthenic acids, zinc salts in petroleum ether and olive oil (4 : 1) through application to the dorsum of both ears for 3 consecutive days. On day 4, 5- bromo-7-deoxyuridine (BrdU) was administered intraperitoneally to each mousse for counting the total cell numbers from each mouse. At the time of lymph node excision, the thickness of the ears was measured at the edge of the right pinna to calculate the ear thickness index. There was an increase of the stimulation index, which was accompanied with an increase in the irritation index, therefore the results are not conclusive about the sensitizing properties of naphthenic acid, zinc salts and naphthenic acids under the conditions of the LLNA test.

 

Since naphthenic acids and its salts seem to have a skin irritation potential, the local lymph node assay is not appropriate to determine the skin sensitisation potential of naphthenic acids or any of tis salts.

 

Two guinea pig maximisation tests in 6-week-old female Hartley guinea pigs were performed with Naphthenic acid, zinc salts and naphthenic acids (Yamano, 2006). The first induction dose was set at 1%, while the second induction dose was 25%. Two weeks after the second induction, 0.1 ml aliquots of the test substance in vehicle (0.5% in petroleum ether) was applied to a shaved area of the flank for challenge. Each site was scored 48 hours after challenge. Naphthenic acid, zinc salts and naphthenic acids resulted in 3/5 and 4/5 animals with clear reactions at 48 hours, respectively. Therefore, both substances were considered to be sensitizing for skin.

 

These results are supported by a QSAR prediction. Since naphthenic acids do not have a fixed composition, the prediction was performed on a broad selection of different possible structures in accordance with the substance identification. As can be seen from the results, almost all of the molecules in Naphthenic acids were identified as being skin sensitisers. Only two molecules were indicated to be a non-sensitiser with a low applicability domain index (ADI). Most molecules fell in the applicability domain (with ADI >=0.8) and thus the prediction can be assumed to be reliable.

 

In contrast to that, in a well performed and guideline compliant guinea pig maximisation test with zinc oxide, no skin reactions were evident in both experimental and control animals (Van Huyenvoort, 1999a). Thus, zinc is not considered to be a skin sensitizer, which is fully in line with the non-classification for skin sensitisation of zinc oxide.

 

Overall, there is a clear evidence that naphthenic acids and its salts do have skin sensitisation potential. However, while both naphthenic acids, zinc salts and naphthenic acids show skin sensitisation potential in appropriate test systems, the information about the potency of the sensitizing effects is not consistent and cannot be appropriately assessed with the given information (animals number in the GPMT too low for an assessment of the potency). Therefore, it is not possible to allocate a subclassification to category 1A (high potency) or 1B (low to moderate potency). Thus, based on a weight of evidence approach and according to the criteria given in Regulation 1272/2008 and its subsequent amendments, naphthenic acids, zinc salts, basic is classified as Skin sensitizer, Cat. 1 H317: May cause an allergic skin reaction.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Respiratory sensitisation:

No animal respiratory sensitisation study with Naphthenic acids, zinc salts, basic is available. However, respiratory sensitisation data in humans are available and reported in section 7.10.4.

A questionnaire was circulated among EU-based manufacturers and downstream usesrs to collect evidence whether Naphthenic acids, zinc salts, basic causes respiratory (and skin) sensitisation. All registrants of the substance in Europe responded to the questionnaire. Currently, a total of 38 workers are involved in the handling and/or processing of the substance in Europe. None of these workers show symptoms of occupational asthma and/or dermal sensitisation. All workers are also handling other substances that are classified as respiratory sensitisers. In total, 143 workers underwent medical surveillance during the last decade, again without any clinical signs of respiratory sensitisation.

Based on the comprehensive information collected in all relevant EU-workplaces, a classification for respiratory sensitisation is not warranted.

Justification for classification or non-classification

Skin sensitisation:

Overall, there is a clear evidence that naphthenic acids and its salts do have skin sensitisation potential. However, while both naphthenic acids, zinc salts and naphthenic acids show skin sensitisation potential in appropriate test systems, the information about the potency of the sensitizing effects is not consistent and cannot be appropriately assessed with the given information (animals number in the GPMT too low for an assessment of the potency). Therefore, it is not possible to allocate a subclassification to category 1A (high potency) or 1B (low to moderate potency). Thus, based on a weight of evidence approach and according to the criteria given in Regulation 1272/2008 and its subsequent amendments, naphthenic acids, zinc salts, basic is classified as Skin sensitizer, Cat. 1 H317: May cause an allergic skin reaction.

 

Resp. sensitisation:

No animal respiratory sensitisation study with Naphthenic acids, zinc salts, basic is available. However, respiratory sensitisation data in humans are available and reported in section 7.10.4.

A questionnaire was developed focussing on the potential of Naphthenic acids, zinc salts, basic to cause respiratory (and skin) sensitisation. None of the exposed workers in Europe reported any sign of respiratory sensitisation in the last year and the last decade. Thus, naphthenic acids, zinc salts, basic is not to be classified according to regulation (EC) 1272/2008 and its subsequent amendments for respiratory sensitisation.