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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Remarks:
(IMDS modification)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 19 March 2012 to 22 March 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study performed similarly to the OECD TG 429, with IMDS modification. Although not a validated method, the IMDS modification is scientifically accepted. A position paper justifying the use of this modified LLNA is attached to this ESR, and summarised in brief within the executive summary section.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010
Deviations:
yes
Remarks:
Modified LLNA (IMDS). Cell proliferation was measured by cell counting instead of radioactive labeling.
Principles of method if other than guideline:
The modified Local Lymph Node Assay (IMDS) was performed on 24 female NMRI mice of the strain Crl:NMRI BR (6 animals/test item group and 6 control animals) to determine if there is any specific (sensitizing) or non-specific (irritant) stimulating potential of the test item Macrolex Blau 3R.
The modifications refer to the measurement of cell proliferation by cell counting instead of radioactive labeling. In addition, the acute inflammatory skin reaction is determined to discriminate specific from non-specific activation of immune competent cells in the draining lymph nodes.
GLP compliance:
yes (incl. QA statement)
Type of study:
other: LLNA (IMDS)

Test material

Constituent 1
Chemical structure
Reference substance name:
1,4-bis[(2-ethyl-6-methylphenyl)amino]anthraquinone
EC Number:
255-460-7
EC Name:
1,4-bis[(2-ethyl-6-methylphenyl)amino]anthraquinone
Cas Number:
41611-76-1
Molecular formula:
C32H30N2O2
IUPAC Name:
1,4-bis[(2-ethyl-6-methylphenyl)amino]-9,10-dihydroanthracene-9,10-dione
Details on test material:
Test item: Macrolex Blau 3R
Synonym: Reinblau RLW
CAS name: 1,4-Bis[(2-ethyl-6-methylphenyl)amino]-9,10-anthracenedione
CAS number: 41611-76-1
Batch No.: CHN23196
Content: 99.5 % (not used for calculation)
Sum formula: C32H30N2O2
Molecular mass: 474.6 g/mol
pH-value: 1:9 diluted in water: 6.3
Physical state: solid
Appearance: blue
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature.
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage:
Nominal value in %: 1 / Content (% of nominal value): 99% at start, 100% after 2 hours, 97% after 4 days.
Nominal value in %: 50 / Content (% of nominal value): 101% at start, 110% after 2 hours, 98% after 4 days.
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: n/a
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: n/a.
- Reactivity of the test material with the incubation material used (e.g. plastic ware): n/a

TREATMENT OF TEST MATERIAL PRIOR TO TESTING :
- Treatment of test material prior to testing (e.g. warming, grinding): The test item was formulation once before application in DMF. The formulations were visually described as suspensions.
- Preliminary purification step (if any): n/a
- Final concentration of a dissolved solid, stock liquid or gel: n/a
- Final preparation of a solid (e.g. stock crystals ground to fine powder using a mortar and pestle): n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material) : n/a

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: pH: 1:9 diluted in water: 6.3

In vivo test system

Test animals

Species:
mouse
Strain:
NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Nederland, Kreuzelweg 53, 5960 AD Horst, Netherlands.
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: n/a
- Age at study initiation: 6 weeks
- Weight at study initiation: 25 - 31 grams
- Housing: During the adaptation period up to 8 mice were housed together in conventional Makrolon® type III cages, While during the study period the animals were single-housed in type II cages.
- Diet (e.g. ad libitum): ad libitum, PROVIMI KLIBA SA 3883 maintenance diet for rats and mice (from Provimi Kliba SA, CH-4303 Kaiseraugst).
The nutritive composition of PROVIMI KLIBA SA 3883 and the contaminant content of the standard diet were checked and analyzed routinely in random samples.
- Water (e.g. ad libitum): tap water (drinking bottles) were provided ad libitum. Polycarbonate bottles with a capacity of about 300 ml (study period) or 700 ml (adaptation period) were used for drinking water. The tap water was of drinking water quality
- Acclimation period: at least 6 days
- Indication of any skin lesions: only healthy animals showing no signs of disease were used in the study

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

- IN-LIFE DATES: From: To: n/a

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
Group 1 Vehicle (DMF)
Group 2 2% Macrolex Blau 3R (in DMF)
Group 3 10% Macrolex Blau 3R (in DMF)
Group 4 50% Macrolex Blau 3R (in DMF)
Group 5 40% Alpha Hexyl Cinnamic Aldehyde (in DMF)
No. of animals per dose:
6 animals/test item group and 6 control animals
Details on study design:
PRE-SCREEN TESTS: n/a

MAIN STUDY:
The methods used in this study are in principle specified in guidelines (OECD 406, 1992; EPA guideline OPPTS 870.2600, Skin Sensitization, March 2003; CPMP/SWP/2145/00, 2001; updated OECD TG 429, 2010). According to these guidelines the so-called Local Lymph Node Assay (LLNA) is recommended for the assessment of skin sensitization as first-stage screening study or as a stand-alone test (updated OECD TG 429; OPPTS 870.2600).
A modification of the assay by measuring the cell counts instead of radioactive labeling provides comparable sensitivity, and has the advantage that the cell suspension can be further analyzed by different methods (flow cytometry, chemiluminescence responses, immunofluorescence) to gain an insight into mechanistic events. A further modification was done by including the measurement of the ear swelling after treatment leading to a much more simplified and reliable assay (Integrated Model for the Differentiation of Skin reactions (IMDS). By comparing the specific immune reaction induced by the test item in the draining lymph nodes (LN; cell counts / LN weights) with the immediate unspecific acute skin reaction (ear swelling / ear weight) it is possible to discriminate the irritant potential from the sensitizing potential of the compound tested. International standards have been successfully determined using this modification. Such modifications are also authorized in the Note of Guidance SWP/2145/00 of the CPMP (2001) and updated OECD guideline 429.

Investigations
- Autopsies
The animals were anaesthetized by inhalation of carbon dioxide and sacrificed one day after the last application (day 4). The appropriate organs were then removed. Lymphatic organs (the auricular lymph nodes) were transferred into physiological saline (PBS).
- LLN Weight and cell count determinations
The weight and cell count determinations were carried out by appropriate laboratory procedures. The weights of the lymph nodes were determined on a Mettler semiautomatic balance and stored in an IBM compatible PC. After crushing the lymph nodes through a sieve in a 12-well plate, the cell counts per ml were determined using a Multisizer 3® from Coulter Electronics. These data were also directly collected and processed by computer (Multisizer 3 software and Excel). Means, indices and standard deviations were calculated by an Excel data sheet. A special BASIC program (GWBASIC compiler) was used to calculate means and standard deviations of the Lymphnodes’ weights. Indices were calculated manually.
The so-called stimulation (or LLN-) index is calculated by dividing the absolute number of weight or cell counts of the substance treated lymph nodes by the vehicle treated ones. Thus, in case of no stimulating effect the index is always about 1.00 (+/- standard deviation), and the indices of vehicle treated animals are set to 1.00 (+/- standard deviation).
- Ear Swelling
Before the first treatment and before sacrifice the thickness of both auricles of the animals was measured using a spring-loaded micrometer (Oditest, Dyer Company or Fa. Kroeplin). Means, indices and standard deviations of the ear swelling were calculated by an Excel data sheet.
- Ear weight
On day 4 of the study the ear weight of the sacrificed animals was measured using a punch to take of a piece of every ear with a diameter of 8 mm. The weights were determined on a Mettler semiautomatic balance. Means, indices and standard deviations of the ear weights were calculated by an Excel data sheet.
- Body weights
The body weights of the animals were recorded at the start and the end of the study (day 1 and day 4)

ANIMAL ASSIGNMENT AND TREATMENT
Six animals were placed in each group. The animals were identified by cage labels giving the test item, the animal number, dose, sex, and the study number and marking of the tail immediately before autopsy.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was formulated, once before administration in DMF. The formulations were visually described as suspensions. respectively. The stability of the test item in the vehicle was analytically verified for up to 4 days.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
When it was statistically reasonable, the values from treated groups were compared with those from the control group(s; vehicle) by a one-way analysis of variance (ANOVA) when the variances are considered homogeneous according to a homogeneity testing like Cochran's test. Alternatively, if the variances are considered to be heterogenous (p~0.05), a non- parametric Kruskal-Wallis test has been used (Kruskal-Wallis ANOVA) at significance levels of 5 %. Two sided multiple test procedures were done according to Dunnett or Bonferroni-Holm, respectively. Outlying values in the LN weights were eliminated at a probability level of 99% by Nalimov's method. In addition, for the LLNA/IMDS the smallest significant differences in the means were calculated by Scheffe's method, which according to Sachs can be used for both equal and unequal sample sizes.
In this method of statistical processing of measurements a large number of comparisons is made, and as a result of the multiple tests the overall probability of error is considerably greater than the p values suggest (increased number of false-positive results). On the other hand, the known methods of adjusting p values lead to an excessive increase in the number of false negatives. In view of these problems the biological and toxicological relevance is also taken into consideration in the evaluation of statistical significance.
For this reason, in the case of indices only the standard deviations between groups and difference analysis ofthe mean values were used in the evaluation ofthe biological relevance.

Results and discussion

Positive control results:
The positive control in the formulation or the vehicle were applied, epicutaneously onto the dorsal part of both ears of the animals. This treatment was repeated on three consecutive days (d1, d2 and d3). A concentration of 40 % was used for Alpha Hexyl Cinnamic Aldehyde as the positive control.
After treatment with HCA, the NMRI mice showed clear increases in the weights of the draining nodes and in the stimulation indices for cell counts compared to control animals, which are of statistically significance. The "positive level", which is 1.4 for cell count indices, has clearly been exceeded.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Remarks:
cell count index
Value:
1
Variability:
+/- 39,31 %
Test group / Remarks:
Group 1 (control)
Key result
Parameter:
SI
Remarks:
cell count index
Value:
0.94
Variability:
+/- 27,50 %
Test group / Remarks:
Group 2 (2%)
Key result
Parameter:
SI
Remarks:
cell count index
Value:
0.97
Variability:
+/- 38,44 %
Test group / Remarks:
Group 3 (10%)
Key result
Parameter:
SI
Remarks:
cell count index
Value:
1.19
Variability:
+/- 24.42 %
Test group / Remarks:
Group 4 (50%)
Key result
Parameter:
SI
Remarks:
cell count index
Value:
1.9
Variability:
+/- 24.42 %
Test group / Remarks:
Group 5 (positive control)
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Result tables are attached to this ESR.

DETAILS ON STIMULATION INDEX CALCULATION
The so-called stimulation (or LLN-) index is calculated by dividing the absolute number of weight or cell counts of the substance treated lymph nodes by the vehicle treated ones. Thus, in case of no stimulating effect the index is always about 1.00 (+/- standard deviation), and the indices of vehicle treated animals are set to 1.00 (+/- standard deviation).

EC3 CALCULATION - not applicable

CLINICAL OBSERVATIONS: None reported

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

SIGNS OF TOXICITY (including dermal irritation at the site of administration, if any, e.g. increased ear thickness): none reported

HISTORICAL CONTROL DATA
The Local Lymph Node Assay Test methodology was checked for reliability in a test on female NMRI mice using Alpha Hexyl Cinnamic Aldehyde formulated in different vehicles (PEG 400, DAE 433, DMF, MEK, acetone/olive oil (4:1) and Cremophor ELl physiological saline solution 2% v/v) at concentrations of 3 %, 10% and 30%. The sensitivity as well as the reliability ofthe experimental technique is thus confirmed by this study. A similar check is done in regular intervals using one ofthe above mentioned vehicles in order to confirm the reliability of the method. The last reliability test using Alpha Hexyl Cinnamic Aldehyde formulated in acetone/olive oil (4:1) at concentrations of 3 %, 10% and 30% clearly showed the sensitizing potential of the test item.

Any other information on results incl. tables

Based on results obtained in validation studies and general experiences with this test system groups of mice were treated with vehicle, 2 %, 10 % or 50 % Macrolex Blau 3R in DMF.


 


The NMRI mice did not show an increase in the stimulation indices for cell counts or for weights of the draining lymph nodes after application of the test item Macrolex Blau 3R.


 


The “positive level”, which is 1.4 for the cell count index, was never reached or exceeded in any dose group.


 


The “positive level” of ear swelling, which is 2x10E-2 mm increase, i.e. about 10 % of the control values, has not been reached or exceeded in any dose group.


No substance specific effects were determined for ear weights either.


 


After treatment with Alpha Hexyl Cinnamic Aldehyde (group 5) the NMRI mice showed clear increases in the weights of the draining lymph nodes and in the stimulation indices for cell counts compared to control animals, which are of statistical significance. The “positive level”, which is 1.4 for cell count indices, has clearly been exceeded.


 


The “positive level” of ear swelling, which is 2x10E-2 mm increase, i.e. about 10 % of the control values, has been exceeded in the positive control group. This increase is of statistical significance. A significant increase compared to vehicle treated animals regarding ear weights was detected, too.


 


It has to be clarified that the “positive levels” mentioned above are exclusively defined for the NMRI outbreed mice used for this study. Such positive limits have to be calculated for each strain of mice individually.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item Macrolex Blau 3R has no sensitizing potential in mice after dermal application of up to and including a 50 % concentration.
Executive summary:

A modified Local Lymph Node Assay (IMDS) was performed on 24 female NMRI mice of the strain HsdWin:NMRI (6 animals/test item group and 6 control animals) to determine if there is any specific (sensitizing) or non-specific (irritant) stimulating potential of the test item Macrolex Blau 3R. A concurrent control of 6 animals treated with Alpha Hexyl Cinnamic Aldehyde was included.


 


Materials and Methods


The study was conducted according to OECD Guidelines No. 429 and No. 406, EC Guideline 2004/73/EC (29th Adaptation of Guideline 67/548/EEC, B.42)/Health Effects Test Guideline and OPPTS 870.2600 (EPA), using the modified LLNA-IMDS approach, with the following test item concentrations:


Test item: 0 % (vehicle control), 2 %, 10 % and 50 %.


Positive control: 40 % Alpha Hexyl Cinnamic Aldehyde


 


The test item was formulated in dimethylformamide (DMF) to yield a suspension.


The positive control was formulated in dimethylformamide (DMF) to yield a solution.


 


The modified LLNA-IMDS (Integrated Model for Differentiation of Skin Reactions)





The standard LLNA has been criticised because it uses radioactive thymidine (which is both limiting as it requires an isotope laboratory, because the i.v. application of [3H] thymidine leads to relatively high individual variance), and due to the incidence of false-positive findings due to irritation and other mechanisms.


The IMDS modification measures cell proliferation by cell counting instead of radioactive labeling. In addition, the acute inflammatory skin reaction is determined to discriminate specific from non-specific activation of immune competent cells in the draining lymph nodes. A position paper has been produced on the modified LLNA (referred to as LLNA-IMDS), and is attached to this endpoint study record (Vohr et al, 2009). 


 





In brief, the modified LLNA-IMDS was investigated in depth in an intra-laboratory validation, published in 2000 (Vohr, HW. et al., 2000). This modified LLNA (as LLNA-IMDS) was subsequently carried out in-house under full GLP conditions as an alternative to guinea pig testing. The reliability of LLNA- IMDS was also demonstrated in CBA mice one year later in an inter-laboratory study (Suda, A. et al., 2001).


As recognised by both ICCVAM and the regulators in the interim, the specific cut-off value (ECt) must be determined for each endpoint to establish cell proliferation and for the murine strain used. The cut-off values are dictated by two parameters: i) individual endpoint variance in the group of individual animals, and ii) the possible maximum stimulation (maximum index) that can be achieved for the relevant parameter. Relatively high stimulation indices (SI) are normally achieved with the radioactive method but the individual variances obtained from individual animal measurements are also rather high. The cut-off that determines a positive result was therefore set at a proliferation index of factor 3 (EC3) for this method. Based on historical LLNA-IMDS data and the assessments of all intra- and inter-laboratory validation studies, the cut-off for the cell count index was set at 1.4 for NMRI (outbred) mice. 


In 2004, the reliability of the modified LLNA (LLNA-IMDS) was investigated in an international catch-up validation study. Extremely convincing results were published in two papers in 2005 (Ehling, G. et al., 2005a; 2005b). In this international study, the cut-off values were confirmed as 1.4 and 1.5 for NMRI (outbred) and BALB/c, respectively. This minor difference is due to the fact that BALB/c mice react slightly more violently overall to non-specific activations – hence the individual variance is somewhat greater than in NMRI (outbred) mice.


 





Based on copious data and detailed rationale, the modified LLNA (LLNA-IMDS) should be considered as reliable, robust and at least as sensitive as the standard LLNA. The IMDS modification of the LLNA has been published several times in various recognised journals. Moreover, the method has been repeatedly described in meticulous detail in these journals. All validation studies were performed under full GLP conditions. The LLNA-IMDS protocol therefore complies with OECD 429 requirements in every respect.


 





An English language copy of the full position paper is attached (Vohr, 2009). 


 


Results


Compared to vehicle-treated animals, none of the parameters measured in the substance-treated groups, i.e. cell counts and weights of the draining lymph nodes, ear weights and ear swelling, reached or exceeded the "positive levels" defined for this assay. These results show that there is no indication for a skin sensitizing effect after administration of a concentration up to and including 50 % Macrolex Blau 3R in this test system.


 


Conclusion


In conclusion, these results show that the test item Macrolex Blau 3R has no sensitizing potential in mice after dermal application of up to and including a 50 % concentration. No indication for a non-specific (irritant) activation was detected, too.  


These results are verified by the comparison with the results of the positive control group (Alpha Hexyl Cinnamic Aldehyde).