Reaction mass of (Z)-1-chloro-1-propene and 2-chloropropane and 2-chloropropene

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according to OECD Guideline n°429

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

2-chloropropene

EC Number:

209-187-5

EC Name:

2-chloropropene

Cas Number:

557-98-2

Molecular formula:

C3H5Cl

IUPAC Name:

2-chloroprop-1-ene

Constituent 2

Reference substance name:

2-chloropropane

EC Number:

200-858-8

EC Name:

2-chloropropane

Cas Number:

75-29-6

Molecular formula:

C3H7Cl

IUPAC Name:

2-chloropropane

Constituent 3

Reference substance name:

1-chloropropene

EC Number:

209-675-8

EC Name:

1-chloropropene

Cas Number:

590-21-6

IUPAC Name:

1-chloroprop-1-ene

Details on test material:

Identification Reaction mass containing mainly 2-chloropropene
Molecular formula C3H5Cl
Molecular weight 76.53
CAS Number 557-98-2
Description Clear light yellow to brown liquid (determined at NOTOX)
Batch RBA100301A
Purity Minimum 60.0%
Test substance storage In refrigerator (2-8°C) in the dark
Stability under storage conditions Stable
Expiry date 08 March 2011 (allocated by NOTOX, 1 year after receipt of the test substance)

Study specific test substance information:
General information Extremely flammable
Test substance handling If necessary, handling after sampling will be performed on dry ice in order to keep the test substance liquefied
Density 0.9
Stability in vehicle:
• Acetone Not indicated
• Olive oil Not indicated
Solubility in vehicle:
• Acetone Not indicated
• Olive oil Not indicated

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Test system
Species Mouse, CBA/J strain, inbred, SPF-Quality.
Recognized by the international guidelines as the recommended test system (e.g. OECD, EC, EPA).
Source: Janvier, Le Genest-Saint-Isle, France
Number of animals 20 females (nulliparous and non-pregnant), five females per group.
Age and bodyweight Young adult animals (approx. 10 weeks old) were selected. Body weight variation was within +/- 20% of the sex mean.
Identification Tail mark with marker pen.
Health inspection A health inspection was performed prior to treatment, to ensure that the animals were in a good state of health. Special attention was paid to the ears, which were intact and free from any abnormality.
Reliability check The results of a reliability test with Alpha-hexylcinnamicaldehyde, performed not more than 6 months previously, are
summarized in the appendix of the report. Similar procedures were used in the reliability test and in this study.

Animal husbandry
Conditions
Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21.0 ± 3.0ºC (actual range: 20.7 – 23.3ºC), a relative humidity of 40-70% (actual range: 42 - 70%) and 12 hours artificial fluorescent light and 12 hours darkness per day.

Accommodation
Individual housing in labeled Macrolon cages (MI type; height 12.5 cm) containing sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) was supplied as cage-enrichment.
The paper was removed on Day 1 prior to dosing and was supplied again after scoring of the ears on Day 3.

Acclimatization period
The acclimatization period was at least 5 days before the start of treatment under laboratory conditions. Accommodation was as described above except that the animals were group housed in Macrolon cages (MIII type; height 18 cm).

Diet
Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).

Water
Free access to tap water.

Results of analysis for diet (nutrients and contaminants), sawdust, paper and water were assessed and did not reveal any findings that were considered to have affected the study integrity. All certificates and results of analysis are retained in the NOTOX archives.

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Three test substance concentrations were tested; a 25%, 50% and 100% concentration (w/w).

No. of animals per dose:

5

Details on study design:

Test substance preparation
Vehicle Acetone/Olive oil (4:1 v/v) (Acetone p.a.: Merck, Darmstadt, Germany; Olive oil: Sigma-Aldrich, Steinheim, Germany).
Rationale The vehicle was selected based on trial formulations performed at NOTOX and on test substance data supplied by the sponsor.
Preparation The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. No adjustment was made for specific gravity of the vehicle. Homogeneity was obtained to visually acceptable levels. Cooled glassware and syringes were used for formulations. If a vehicle was used: Refrigerated vehicle was used. The formulation was kept in a bath with crushed ice until dosing. If dosed undiluted: The test substance was kept in a bath with crushed ice until dosing.
Preliminary irritation study:
A preliminary irritation study was conducted in order to select the highest test substance concentration to be used in the main study. In principle, this concentration should be well tolerated systemically by the animals and may give moderate irritation (maximally grade 2; see section 6.6) at the highest concentration.

Two test substance concentrations were tested; a 50% and 100% concentration. The highest concentration was the maximum concentration as required in the test guidelines (undiluted for liquids). The test substance and formulation were kept in a bath with crushed ice during dosing.
The test system, procedures and techniques were identical to those used during Days 1 to 3 of the main study unless otherwise specified. Two young adult animals were selected (Source: Harlan, Horst, The Netherlands; Age: 8-14 weeks old). Each animal was treated with one concentration on three consecutive days. Approximately 3-4 hours after the last exposure, the irritation of the ears was assessed. Bodyweights were determined on Day 3. The animals were sacrificed after the final observation and no necropsy was performed.

Main study:
Three groups of five animals were treated with one test substance concentration per group. The highest test substance concentration was selected from the preliminary irritation study.
One group of five animals was treated with vehicle.
The test substance and formulations were kept in a bath with crushed ice during dosing.

Allocation:
Group1 animal numbers induction (test substance; % w/w)

1 01 - 05 0 (Acetone/Olive oil (4:1 v/v))
2 06 - 10 25
3 11 - 15 50
4 16 - 20 100

1. five females each group

Induction - Days 1, 2 and 3:
The dorsal surface of both ears was epidermally treated (25 μL/ear) with the test substance concentration, at approximately the same time per day. The concentrations were mixed thoroughly using a vortex mixer immediately prior to dosing. The test substance and formulations were kept in a bath with crushed ice during dosing.

The control animals were treated the same as the experimental animals, except that the vehicle was administered instead of the test substance.

Excision of the nodes - Day 6:
All animals:
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 μCi of 3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US).

After approximately five hours, all animals were killed by intraperitoneal injection with Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). The draining (auricular) lymph node of each ear was excised.
The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 mL PBS.

Tissue processing for radioactivity - Day 6:
A single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter 125 μm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) and stored in the refrigerator until the next day.

Radioactivity measurements - Day 7:
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactive measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

Observations:
Mortality/Viability Twice daily.

Toxicity At least once daily.

Body weights On Days 1 (pre-treatment) and 6.

Necropsy No necropsy was performed according to protocol.

Irritation On Day 3 (3-4 hours after treatment), the skin reactions were assessed. Skin reactions were graded according to the following numerical scoring system. Furthermore descriptions of all other (local) effects were recorded.

Positive control substance(s):

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

Results and discussion

Positive control results:

The SI values calculated for the substance concentrations 5, 10 and 25% were 1.7, 2.7 and 8.8 respectively. An EC3 value of 10.7% was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range of 2 and 20%. The results of the 6 monthly HCA reliability checks in CBA/J female mice of the recent years were 14.1, 13.8, 13.9, 16.0, 11.9 and 16.9%.
Based on the results, it was concluded that the Local Lymph Node Assay in the mouse as supplied by Janvier performed at NOTOX is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: The SI values calculated for the substance concentrations 25, 50 and 100 % were 1.1, 0.8 and 0.9 respectively.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 25, 50 and 100 % were 734, 534 and 601 DPM respectively. The mean DPM/animal value for the vehicle control group was 644 DPM.

Any other information on results incl. tables

Preliminary irritation study (Table 1)

The results of the epidermal exposures for the selection of highest test substance concentration to be tested in the main study are presented in the table.

Based on these results, the highest test substance concentration selected for the main study was a 100% concentration.

 

Main Study

Skin reactions / Irritation (Table 2)

The slight erythema of the ears as observed among animals at 50% and for all animals at 100% was considered not to have a toxicologically significant effect on the activity of the nodes. No oedema was observed in any of the animals examined.

 

Macroscopy of the auricular lymph nodes and surrounding area (Table 2)  

All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size.

No macroscopic abnormalities of the surrounding area were noted in any of the animals.

 

Body weight (Table 2)

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The slight body weight loss, noted in some animals, was considered not toxicologically significant.

 

Radioactivity measurements (Tables 3 and 4)

Mean DPM/animal values for the experimental groups treated with test substance concentrations 25, 50 and 100% were 734, 534 and 601 DPM respectively.

The mean DPM/animal value for the vehicle control group was 644 DPM.

 

Toxicity and mortality 

No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

 

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

The SI values calculated for the substance concentrations 25, 50 and 100% were 1.1, 0.8 and 0.9 respectively (Table 4).

Since there was no indication that the test substance elicit an SI ≥ 3 when tested up to 100%, Reaction mass containing mainly 2-chloropropene was considered to be no skin sensitizer.

The six-month reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity.

Based on these results Reaction mass containing mainly 2-chloropropene would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. It does not have to be classified and has no obligatory labeling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations (2007) and the Regulation (EC) No 1272/2008 on classification, labeling and packaging of substances and mixtures.

Executive summary:

The study was carried out based on the guidelines described in:

-OECD, Section 4, Health Effects, No.429 (2002),

-EC, No 440/2008; B42: "Skin Sensitization: Local Lymph Node Assay"

-EPA, OPPTS 870.2600 (2003) “Skin Sensitization”.

 

Test substance concentrations selected for the main study were based on the results of a preliminary study.

 

In the main study, three experimental groups of five female/J mice were treated with test substance concentrations of 25, 50 or 100% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Acetone/Olive oil (4:1 v/v)).

 

Three days after the last exposure, all animals were injected with³H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal.

After precipitating theof the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of Disintegrations Per Minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

 

The slight erythema of the ears as observed among animals at 50% and for all animals at 100% was considered not to have a toxicologically significant effect on the activity of the nodes. No oedema was observed in any of the animals examined.

 

All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size.

No macroscopic abnormalities of the surrounding area were noted in any of the animals.

 

Mean DPM/animal values for the experimental groups treated with test substance concentrations 25, 50 and 100% were 734, 534 and 601 DPM respectively. The mean DPM/animal value for the vehicle control group was 644 DPM.

 

The SI values calculated for the substance concentrations 25, 50 and 100% were 1.1, 0.8 and 0.9 respectively (Table 4 and Figure 1).

 

Since there was no indication that the test substance elicit an SI≥3 when tested up to 100%,Reaction mass containing mainly 2-chloropropenewas considered to be no skin sensitizer.

 

The six-month reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity.

 

Based on these results Reaction mass containing mainly 2-chloropropene would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. It does not have to be classified and has no obligatory labeling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations (2007) and theRegulation (EC) No 1272/2008 on classification, labeling and packaging of substances and mixtures.