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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 09 to June 06, 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study according to OECD Guideline 429; Study under GLP Regulation;
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan, NL-5960 AD Horst, The Netherlands
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 16-24 g
- Housing:cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period:under test conditions
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0.0% (Control), 5, 10, 25, 50, 100% (w/v)
No. of animals per dose:
4 female animals per dose
Details on study design:
RANGE FINDING TESTS:
- Lymph node proliferation response: at 10, 25,50 and 100% (w/v)
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
As positive control for the current LLNA test, three groups each of four female mice were treated daily with alpha-hexylcinnamaldehyde at concentrations of 5 %, 10 % and 25 % (w/v) in acetone:olive oil, 4:1 (v/v) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. A control group of four mice was treated with the vehicle (acetone:olive oil, 4:1 (v/v).

The results obtained (STIMULATION INDEX (S.I.) for this positive test are reported as follows:.

Group 2 5* % (w/v) 2.4 * (S.I.)
Group 3 10 * % (w/v) 3.6 * (S.I.)
Group 4 25 % (w/v) 11.2 (S.I.)

A clear dose-response relationship was observed. * This value was used in calculation of EC3. The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value and found to be EC3 = 7.5 % (w/v),
Parameter:
SI
Remarks on result:
other: 0.6, minimum 1.0, maximum
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: 4'890 minimum; 2'589 maximum;

Results

              

Calculation and results of individual data

The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured on a 13-scintillation counter See in text table below for stimulation index (S.I.).

Test item concentration

% (w/v)

S.I.

Group 2

5

0.6

Group 3

10

0.6

Group 4

25

1.0

Group 5

50

0.6

Group 6

100 (undiluted)

0.5

No dose-response relationship was observed.

Calculation of the EC 3 value was not performed because no test concentrations produced a STIMULATION INDEX (S.I.) of 3 or higher.

The radioactive disintegration values for the individual treatment groups are included in Table 1 below.

Viability / Mortality

No deaths occurred during the study period.

Clinical Signs

No clinical signs were observed in any animals of the control group, Group 2 (5 %), Group 3 (10 %) or Group 4 (25 %). On the second application day, a slight to moderate ear erythema was observed at both dosing sites in all mice of Group 5 (50 %) and Group 6 (100 %,  undiluted), persisting for a total of two days. The individual clinical were graded in severity of the symptoms into four grades: slight (1), moderate (2), severe (3) and very severe (4),.

Body weights

 

The body weight of the animals, recorded prior to the first application and prior to necropsy, was within the range commonly recorded for animals of the strain and age.

Table 1

Test item

Measurement

Calculation

Result

concentration

dpm

dpm -

number of

dpm per

S.I.

% (w/v)

BGa)

lymph nodes

lymph nodeb)

--

BG I

10

--

--

--

--

--

BGII

14

--

--

--

--

--

CG 1

4902

4890

8

611

--

5

TG 2

2978

2966

8

371

0.6

10

TG 3

2898

2886

8

361

0.6

25

TG4

4846

4834

8

604

1.0

50

TG5

2726

2714

8 **

339

0.6

100 *

TG6

2601

2589

8 **

324

0.5

*, undiluted as delivered by sponsor;

**, the size of the draining lymph nodes of this group was obviously small compared to those of the control group;

BG, background (1 ml 5% trichloroacetic acid) in duplicate;

CG, control group;

TG, test group;

S.I., simulation index

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the STIMULATION INDEX (S.I.).

RHODIASOLV RPDE was found to be a non-sensitizer when tested at up to the concentration of 100 % (undiluted).

Executive summary:

In order to study a possible contact allergenic potential of RHODIASOLV RPDE,  a local lymph node assay (LLNA) was performed according to the OECD Guideline 429 and und GLP Regulations. Five groups each of four female mice were treated daily with the test item at concentrations of 5 %, 10 %, 25 %, 50 % (w/v) in acetone/olive oil (4/1, v/v) and 100 % (undiluted) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. A control group of four mice was treated with the vehicle (acetone/olive oil (4/1, v/v)) 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. All treated animals survived the scheduled study period. No clinical signs were observed in any animals of the control group, Group 2 (5 %), Group 3 (10 %) or Group 4 (25 %). On the second application day, a slight to moderate ear erythema was observed at both dosing sites in all mice of Group 5 (50 %) and Group 6 (100 %, undiluted), persisting for a total of two days.

The results obtained (Stimulation Index (S.I.)) for each group of animals and exposure were as follows: group 2 (5% (w/v)), S.I. 0.6; group 3 (10% (w/v)), S.I. 0.6; group 4 (25% (w/v)), S.I. 1.0; group 5 (50% (w/v)), S.I.  0.6; group 6 (100% (w/v)), S.I. 0.5.

A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the stimulation index (S.I.). In this study S.I. of 0.6, 0.6, 1.0, 0.6 and 0.5 were determined with the test item at concentrations of 5 %, 10 %, 25 %, 50 % (w/v) in acetone/olive oil (4/1, v/v) and 100 % (undiluted), respectively.

RHODIASOLV RPDE was therefore found to be a non-sensitizer when tested at up to the concentration of 100 % (i.e., undiluted).

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

The skin sensitization potential of dibasic esters was assessed in two in vivo assays. A mouse Local Lymph Node Assay (LLNA), of reliability 1 according to Klimisch cotation criteria, was selected as a key study based on its high reliability and as it is the reference method endorsed by REACH. Several concentration of dibasic esters blend were tested up to 100%, and no increase in the stimulation indices occurred, illustrating the absence of skin sensitization. This is confirmed by a Guinea-Pig Maximisation Test (GPMT), of reliability 2 according to Klimisch cotation criteria and therefore selected as a supporting study, where the concentrations of 10% and 100% were tested. No cutaneous reactions were observed following challenge, corroborating the absence of skin sensitization potential.


Migrated from Short description of key information:
No indication of skin sensitization potential in a Local Lymph Node Assay and a Guinea Pig Maximisation Test.

Justification for selection of skin sensitisation endpoint:
The LLNA was selected as a key study based on its high reliability and as it is the reference method endorsed by REACH

Respiratory sensitisation

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

The respiratory sensitization potential of dibasic esters was not formally assessed in a dedicated study, but there was no indication of specific immunotoxicity in rats exposed by inhalation for up to 90 days (see "7.5. Repeated dose toxicity" section for details).

 

Therefore dibasic ester blend is not considered to be a respiratory sensitizer.


Migrated from Short description of key information:
Not formally assessed but no indication of specific immunotoxicity in 90-day inhalation toxicity studies in rats.

Justification for classification or non-classification

Based on the classification criteria of Annex VI Directive 67/548/EEC or EU Regulation 1272/2008 (CLP), and given the absence of positive reactions in a LLNA and a GPMT, or signs of specific immunotoxicity in a rat 90 -day toxicity study by inhalation, dibasic ester blend is not classified as a skin or respiratory sensitizer.