Distillation residue, butyl alcohols production, rectification

Endpoint summary

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:

2010-08-10 to 2010-09-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was performed in compliance with OECD TG429 Skin sensitization: local lymph node assay, following Good Laboratory Practice regulations.

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Animals
Species: Mice 18-20g (Delivery protocol no. 18/2010)
Strain: CBA/Ca
Source: Velaz s.r.o., Lysolaje 15, 165 00 Praha 6
Number and Sex:
5 females – control
5 females – positive control
20 females – test substance

Identification: The animals (8-12 weeks old, 18-20 g) were housed individually in plastic cages TouchSLIMPlus apparatus (Trigon s.r.o.). The animals were marked in groups by serial numbers 1 - 5. These numbers were placed on the cage together with the marking of group.

Housing: The mice were housed according to SOPB-00187-AH Laboratory Mouse and Government order no. 23/2009.

Diet: The standard diet MP (TOP DOVO) was served. The supplier of the diet is approved by State Veterinary and Food Administration SR

Water: Ad libitum.

Environment: Environmental controls for the animal room were set to maintain 22 ± 2oC, a relative humidity of 55 ± 10 % a minimum of 10 air changes/hour, and a regulated light regime- 12/12 (SOPA-00141-AH Climatic condition and animal holding in animal house).

Acclimation: According SOPB-00188-AH Animals quarantine .

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

Dose Selection: The doses were selected from the concentration series 100%, 50%, 25%, 10%, 5%, 2.5% etc. according to OECD Guideline 429.

Dose Administration: The test substance was applicated at concentrations of 10, 25, 50 and 100% in volume of 25 microliters to the dorsum of each ear. The alpha-Hexylcinnamaldehyde (25%) as positive control and the vehiculum as a control were used in the same volume.

The test substance delivered by sponsor was considered as 100% solution.

The test substance and positive control solutions were prepared daily before application.

Vehiculum: Acetone/olive oil (4:1 v/v)

No. of animals per dose:

5 females - control
5 females - positive control
20 females - test substance

Details on study design:

Test procedure
Day 1:
Each animal was identified and the body weight was recorded (SOPA-000072-GEN ). To the dorsum of each ear was treated with 25µl of the appropriate dilution of the test substance, alpha-Hexylcinnamaldehyde or the vehicle alone.
Days 2 and 3:
The application procedure carried out on day 1 was repeated.
Days 4 and 5 :
No treatment.
Day 6 :
The body weight of each animal was recorded. 250µl of phosphate-buffered saline (PBS) containing 20 µCi of 3H-methyl thymidine was injected into all test and control mice via the tail vein.

Five hours later, the animals were killed. The draining auricular lymph nodes from each ear were excised and pooled in PBS for each experimental group (pooled treatment group approach) and weighted.

Clinical observations
Animals were carefully observed once daily for any clinical symptoms, either of local irritation at the application site or of systemic toxicity. All observations were systematically recorded with individual records being maintained for each animal.
The clinical observation were scored as 0 (no effect), + (weak effect), ++ (moderate effect), +++ (strong effect).

Preparation of cell suspensions
Cell suspension of lymph node cells from pooled treatment groups was prepared by gentle mechanical desagregation in glass homogenizer . Lymph node cells were washed with an excess of PBS and centrifuged (SOPA-00156-PH) by 600 g at 40C for 10 min. Suspension of cells were precipitated with 5% trichloroacetic acid (TCA) at 4C degrees for 18-20h. Pellets were centrifuged by 2000g at 40C for 5 min., re-suspended in 1 ml TCA and transferred to scintillation vials containing 10 ml of scintillation fluid for 3H-counting.

Determination of cell proliferation (incorporated radioactivity)
Incorporation of 3H-methyl thymidine into the lymph node cells was measured by oscintillation counting on Liquid scintillation analyzer TRI-CARB, 2000CA, Packard (SOPB-00192-PH) as disintegrations per minute (DPM). The incorporation was expressed as DPM/treatment group .

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Calculation of SI
The SI value was obtained by dividing the pooled radioactive incorporation for each treatment group by the incorporation of the pooled vehicle control group.
The concentration eliciting a SI of 3 is identified as EC3 value and was calculated by linear interpolation of points on the dose-response curve, immediately above and below threefold the threshold having the coordinates (a,b) and (c,d) according to the equation (12):
EC3 = c + [(3-d)/(b-d)] x (a-c).

Parameter:

SI

Remarks on result:

other: The SI values for treated groups were 2.00 (10%), 2.78 (25%), 3.11 (50%) and 3.77 (100%). The calculated EC3 value was 41.75%. The lymph node weights, DPM and SI values are shown in attached table.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

Lymph node proliferation: In comparison with the control group of animals after application of the registered substance, the increase of the pooled lymph node weights was registered in all tested concentrations. This increase was dose dependent. The pooled lymph node weights of treated groups were 0.0308g for 10% concentration, 0.0320g for 25% concentration, 0.0355g for 50% concentration and 0.449g for 100% concentration of tested substance. The lymph node weight of control group was 0.0278g and for positive control group was 0.0588g. The DPM values for treated groups were 1584.35 (10%), 2147.18 (25%), 2399.69 (50%) and 2909.37 (100%).

	Lymph node
	weiht (g)	DPM	SI	EC3
Control	0.0278	772.02
Pos. Control	0.0588	3915.76	5.07
Solvent-still residue 10%	0.0308	1548.35	2	41.75
Solvent-still residue 25%	0.032	2147.18	2.78
Solvent-still residue 50%	0.0355	2399.69	3.11
Solvent-still residue 100%	0.0449	2909.37	3.77

Interpretation of results:

sensitising

Remarks:

Migrated information class 4, rare allergen Criteria used for interpretation of results: EU

Conclusions:

The skin sensitization potency of the target substance was evaluated using LLNA test. SI values over 3 was determined at test substance concentrations of 50 and 100%. Based on the results of this study, we can presume that the substance can be classified as a skin sensitiser.

Executive summary:

The skin sensitization potency of the substance was evaluated by LLNA method, which was developed on the basis of scientific understanding of the immunological mechanism of skin sensitization. In experiments with application of the substance over three consecutive days and at four concentrations the increasing in lymph node weight and DPM was observed in all treated groups and it was dose-dependent. SI values over 3 was determined at concentrations 50 and 100% . In general, when the SI for any single treatment dose group is ≥3, the test substance is regarded as a potential skin sensitizer.

According to EC3 value (41.75) calculated for Solvent-still residue of butyl alcohol rectification and in compliance with the human classes of sensitising potency (the substance was categorised in class 4, as a rare allergen like ethylenglycol dimethacrylate, isopropyl myristate etc. Based on the results of this study, EC3 value and human potency estimation classification(Basketter, D.A. et al. Contact Dermatitis.2000, 42, 344-348), we can presume that the substance LLNA test at used concentrations can be classified as a rare allergen.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The skin sensitization potency of the substance was evaluated by LLNA method which was developed on the basis of scientific understanding of the immunological mechanism of skin sensitization. In experiments with application of the target UVCB-substance over three consecutive days and at four concentrations the increasing in lymph node weight and DPM was observed in all treated groups and it was dose-dependent. SI value over 3 was determined at test item concentrations of 50 and 100 %. In general, when the SI for any single treatment dose group is ≥3, the test substance is regarded as a potential skin sensitizer. Based on the results of the study performed with target substance, we can presume that the substance can be classified as a class 4, rare allergen.

Migrated from Short description of key information:
Study with target UVCB-substance was performed in compliance with OECD TG429 Skin sensitization: local lymph node assay, following Good Laboratory Practice regulations. Based on the study results the test item is regarded as a potential skin sensitiser.

Justification for selection of skin sensitisation endpoint:
The study was conducted in compliance with the OECD 429 guideline and in accordance with GLP.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Migrated from Short description of key information:
There is no information available which would indicate respiratory sensitisation properties.

Justification for classification or non-classification

The target substance is classified for skin sensitization (Skin Sens. 1) according to the CLP Regulation No. 1272/2008 and as R43 according to EU Directive 67/548/EEC.