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Diss Factsheets

Administrative data

Description of key information

A reliable experimental study with the test substance is available (Armondi et al, 1990; GPMT; K1). Due to ambiguous results, this study cannot be used to conclude on the skin sensitization potential of the substance but is considered in a weight of evidence approach together with a reliable in vivo study with the analogue substance NEM (LLNA with NEM, 1997). Based on available results, the test substance is considered to not be skin sensitising. Since the substance is known to be Corrosive to the skin, and in accordance with the 3R principle, no new (in vivo or in vitro) testing should be conducted.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
from 1996-11-14 to 1997-09-19
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Justification for type of information:
Well, but not completely -documented GLP study, following no specific guideline, but similar to OECD guideline 429. Limited test item information is available.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: CP5337 4Y506
Species:
mouse
Strain:
Balb/c
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River
- Age: 9 - 11 weeks
- Weight at study initiation: no data
- Housing: single housing, in wire bottom cages suspended above catch pans containing absorbent cageboard. The cages were sanitized and cage boards changed regularly in accordance with good husbandry practices.
- Diet: Purina Certified Rodent Chow, ad libitum
- Water (e.g. ad libitum): from water nipple attached to automatic water line, ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 1°C
- Humidity (%): 50 ±10%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
30% vol/vol
No. of animals per dose:
5 animals per dose group
Details on study design:
RANGE FINDING TESTS:
Each amine catalyst was tested in the LLNA at the minimal irritating concentration (MIC) determined in previous Primary Dermal Irritation Studies (PDIS). For the amine catalysts where an EC10 could not be calculated, the materials were tested at the highest concentration tested which did not trigger an increase in ear swelling.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
This study consisted of four separate trials. Each trial contained
- a vehicle control group (5 mice treated with a solution of 4: 1 acetone:olive oil, AOO),
- three positive control groups (5 mice treated with a solution of each 1.0% dinitrochlorobenzene (DNCB), 0.17% benzalkonium chloride (BC), and 25% trimellitic anhydride (TMA), and
- five treatment groups (5 mice treated with solutions of an amine catalyst in AOO).

TREATMENT PREPARATION AND ADMINISTRATION:
-Preparation of Dosing Solutions:
The dosing solutions were prepared a maximum of 7 days prior to each test. The vehicle was prepared by mixing four parts of acetone with one part of olive oil (AOO). For the remaining solutions, the appropriate amount of material was weighed into a 16 x 100 mm glass, screw top test tube and the appropriate amount of AOO solution added using a glass pipet (wt/vol.). These solutions were diluted as necessary to obtain the desired concentration (vol. /vol.).

- Topical application:
Prior to the application of the solutions on Day I, an erythema score was assessed for each mouse. The mice were exposed to methoxyflurane. After loosing consciousness, the solutions were applied by dispensing 12.5 ul of the appropriate solution onto the dorsal and ventral side of each ear using an automatic pipettor. Each ear received a total of 25 ul of solution for a total of 50 ul per mouse. The solutions were applied once daily for three consecutive days. On the fourth day, the mice were not treated, but were observed in their cages.

EXCISION OF THE NODES
On Day 5, prior to injection with 3H-thymidine, the erythema score was assessed for each mouse. Each mouse was then injected, via the lateral tail vein, with 0.2 ml of phosphate buffered saline (PBS) containing 20 uCi of 3H-thymidine. Five hours after the injections, the mice were sacrificed by CO2 inhalation and the draining auricular lymph nodes were excised and pooled for each mouse.

TISSUE PROCESSING FOR RADIOACTIVITY
A single cell suspension of lymph node cells (LNC) was prepared as follows:
Both lymph nodes from each mouse were placed in a centrifuge tube containing PBS. This procedure was continued for each mouse. The nodes and PBS were transferred to a small petri dish and dissociated by grinding with the rough end of a syringe plunger. The resulting material was triturated
and the capsule was discarded. The lymph node cells were transferred to a labeled centrifuge tube containing PBS. This process was completed for each liet of lymph nodes. The cell suspensions were centrifuged at 200 x g at 4 °C for 10 minutes. The supernatant was carefully poured off and additional PBS was added to each tube. The tubes were inverted gently and centrifuged again at 200 x g at 4 °C for an additional 10 minutes. The supernatant was carefully poured off and 5% Trichloroacetic acid (TCA) was added to each tube. The tubes were refrigerated at 4 °C for approximately 68 hours.

RADIOACTIVITY MEASUREMENTS
The cell suspensions were centrifuged at 200 x g at 4 °C for 10 minutes and resuspended in 1 ml of 5% TCA. The resuspended cells were transferred to labeled scintillation vials containing 5 ml of Aquasol(R) scintillation cocktail. Each sample was counted for 5 minutes using a beta-scintillation counter.
Radioisotope incorporation was measured as disintegrations per minute (dpm) per mouse.

Interpretation of Raw Data:
A mean dpm value ± SE (standard error) was calculated for each experimental group.
In addition, a stimulation index (SI) was calculated using absolute dpm value for each mouse as the numerator, and the mean dpm value from the vehicle control mice as the denominator. A mean SI ± SE was calculated for each experimental group.

Criteria used to consider a positive response:
Historically, any chemical that produces a SI of> 3 in the LLNA is considered "positive" (Kimber et al., 1994)
Positive control substance(s):
other: 1.0% dinitrochlorobenzene (DNCB)... (see attached file)
Statistics:
mean dpm value ± SE (standard error) and mean SI ± SE was calculated for each experimental group where SI = dpm of mouse / mean dpm of vehicle group
Positive control results:
Trial 2:
Control DNCB: 1.0 %, SI 74.16 ± 7.20
Control BC: 0.17%, SI 4.77 ± 1.19
Control TMA: 25%, SI 72.55 ± 7.65

Trial 4:
Control DNCB: 1.0 %, SI 40.90 ± 3.89
Control BC: 0.17%, SI 3.28 ± 0.45
Control TMA: 25%, SI 41.07 ± 5.27
Key result
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: not reported
Key result
Parameter:
SI
Value:
1.18
Variability:
+/-0.15
Test group / Remarks:
30%
Key result
Parameter:
SI
Value:
1.34
Variability:
+/-0.25
Test group / Remarks:
30%

Observations

-Viability / mortality: no data

- Clinical signs: no data

- Erythema results:

Trial 2 on 1996-11-14

Control DNCB: erythema score 1.0 (day 5), conc. 1.0%

Control BC: erythema score 0 (day 5), conc. 0.17%

Control TMA: erythema score 2.0 (day 5), conc. 25%

CAS 100 -74 -3: erythema score 0 (day 5), conc. 30%

Trial 4 on 1997-02-10

Control DNCB: erythema score 3.0 (day 5), conc. 1.0%

Control BC: erythema score 1.8 (day 5), conc. 0.17%

Control TMA: erythema score 3.0 (day 5), conc. 25%

CAS 100 -74 -3: erythema score 0 (day 5), conc. 30%

Interpretation of results:
GHS criteria not met
Conclusions:
The test item elicited an SI value <3 and is concluded not be a skin sensitizer under the described conditions. Therefore, CLP classification is considered not to be appropriate.
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1990-10-23 - 1990-11-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
Buehler test
Justification for non-LLNA method:
The Murine Local Lymph Node Assay (LLNA) is the first-choice method for in vivo testing according to the REACH Regulation. However, this test was performed before entry into force of the REACH Regulation.
Specific details on test material used for the study:
- Name of test material (as cited in study report): 6398-24-20, Project #90-005
- Substance type: clear colorless liquid
- Physical state: liquid
- Purity: responsibility of the sponsor
- Stability under test conditions: There was no apparent change in the physical appearance of the test article during administration.
Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BuckberG Lab Animals, Tomkins Cove, New York, USA
- Weight at study initiation: 300 - 500 g
- Housing: Guinea pigs were housed individually in stainless steel 1/2 "wire mesh cages sized in accordance with "Guide for the Care and Use of Laboratory Animals" or the Institute of Laboratory Animal Resources, National Research Council.
- Diet (e.g. ad libitum): Purina Guinea Pig Diet, ad libitum. Food was checked daily and added or replaced as needed. Feeders are designed to reduce soiling, bridging and scattering.
- Water (e.g. ad libitum): Fresh tap water, ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30 to 70%
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark
Route:
epicutaneous, occlusive
Vehicle:
other: 80% ethanol (ETOH) - three inductions; acetone - challenge and rechallenge
Concentration / amount:
induction and challenge: 25%
rechallenge: 10%
Route:
epicutaneous, occlusive
Vehicle:
other: 80% ethanol (ETOH) - three inductions; acetone - challenge and rechallenge
Concentration / amount:
induction and challenge: 25%
rechallenge: 10%
No. of animals per dose:
Dose range: 4
Test article: 20
Positive control: 5
Negative control: 10
Naive-rechallenge: 10
Details on study design:
RANGE FINDING TESTS: Prior to initiation of the study, the irritation potential was determined. Four unexposed animals (2 males and 2 females) were each exposed to four different concentrations of the test material using 80% ethanol as the vehicle by the technique described in site preparation and treatment. In this test, both sides of the animal were shaved and exposed to four concentrations of the material. The highest non-irritating concentration was that concentration in vehicle that induced responses not exceeding two + and two 0 grades in the group of four animals. Based upon the results of the dose-range-finding study and in discussion with the sponsor, the dose chosen for the inducation and challenge was 25%. The dose chosen for rechallenge was 10%.

MAIN STUDY
A. INDUCTION EXPOSURE
The left shoulder of each animal was clipped free of hair twenty-four hours prior to the 1st, 2nd and 3rd applications of the test material. The clipped area was approximately 5 x 10 cm, i.e. 10% of the body surface. The test material was applied to 20 guinea pigs (10 males and 10 females). The material was applied beneath a 25 mm hill top chamber and covered with a dental dam. The patch and dam were held in place with clips attached to the sides of the guinea pig restrainer. The patches were allowed to remain in place for six hours, after which the rubber dams and patches were removed. The treated sites were examined after each dosing day and scored at 24 and 48 hours. This procedure was performed once a week for three weeks, a total of three six-hour inductions. A concurrent positive control group consisting of five guinea pigs (3 males and 2 females) was treated with (0.3%) 1-chloro-2,4-dinitrobenzene (DNCB) in 80% ethanol (80:20 ethanol/deionized water). An additional group of ten guinea pigs (5 males and 5 females) was treated with the vehicle.

B. CHALLENGE EXPOSURE
Fourteen days after the last induction sensitizing exposure, the animals were challenged in the same manner on a naive site on the left side. Seven days following the primary challenge, all test article-treated animals, along with an additional group of naive guinea pigs were rechallenged at a 10% concentration. All animals were observed for local (dermal) and systemic effects. Twenty-four hours after the challenge and rechallenge, all animals were depilated with Neet Cream Hair Remover. The depillatory was placed on the test sites surrounding areas and left on for no more than 30 minutes. A minimum of two hours after depliation test sites were graded. The grading was repeated 24 hours later (48 hour grade).

Challenge controls:
The negative control group was challenged with vehicle on the left flank and test article on the right flank.
Positive control substance(s):
yes
Remarks:
1-chloro-2,4-dinitrobenzene
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
slightly patchy erythema only in 5 animals
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25%
No. with + reactions:
4
Total no. in group:
20
Clinical observations:
3 with slightly patchy erythema only and 4 with slight or confluent or patchy erythema
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
0.3%
No. with + reactions:
5
Total no. in group:
5
Clinical observations:
severe erythema with/without edema
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
0.3%
No. with + reactions:
5
Total no. in group:
5
Clinical observations:
severe erythema with/without edema
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
100% aceton
No. with + reactions:
0
Total no. in group:
9
Clinical observations:
no reactions; one animal died
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
100% acetone
No. with + reactions:
0
Total no. in group:
9
Clinical observations:
no reactions; one animal died
Key result
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
10%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
slightly patchy erythema in 3 animals
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
10%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
slightly patchy erythema in 1 animal
Key result
Reading:
rechallenge
Hours after challenge:
24
Group:
other: naive control
Dose level:
10%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no reactions
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
other: naive control
Dose level:
10%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no reactions

One animal died during the course of the study. Necropsy of this animal revealed excessive vascularization of the stomach, intestines and cecum. The death of this animal was considered not to be related to treatment.

Interpretation of results:
study cannot be used for classification
Conclusions:
Based upon the observations made in the Delayed Contact Hypersensitivity Study in Guinea Pigs, the substance induced and challenged at a 25% concentration produced delayed contact hypersensitivity. When rechallenged at a 10% concentration, the substance did not cause delayed contact hypersensitivity in guinea pigs.
Based on the results available, there is no sufficient evidence to be able to conclude on the classification of the substance.
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Justification for type of information:
A read-across approach is followed with the related substance N-ethylmorpholine. Justification of this approach is included in section 13.
Reason / purpose for cross-reference:
read-across source
Key result
Parameter:
SI
Value:
1.34
Variability:
+/-0.25
Test group / Remarks:
30%
Key result
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: not reported
Key result
Parameter:
SI
Value:
1.18
Variability:
+/-0.15
Test group / Remarks:
30%
Interpretation of results:
GHS criteria not met
Conclusions:
The results of in vivo skin sensitisation testing with the related substance N-ethylmorpholine are considered. This source substance elicited an SI value of <3 and was concluded not to be a skin sensitiser under the described conditions. Therefore, CLP classification is considered not to be appropriate. The same is assumed for the target substance .
Endpoint:
skin sensitisation: in vivo (LLNA)
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the study does not need to be conducted because the substance is classified as skin corrosion (Category 1, 1A, 1B or 1C)
Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the study does not need to be conducted because the substance is classified as skin corrosion (Category 1, 1A, 1B or 1C)
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Based on the fact the substance is known to be skin corrosive, no in vitro or in vivo skin sensitisation testing has to be conducted.


The following studies are considered in a weight-of-evidence approach to evaluate  the skin sensitization potential of the test substance:


• Armondi (1990) investigated the sensitising properties of the test substance in a Buehler test with male and female Hartley guinea pigs. The study examined the skin sensitising effect on guinea pigs, using 20 animals in total. Positive, solvent and negative controls were included in the study. Animals were induced and challenged with 25.0% test substance. Based upon the observations made in the Delayed Contact Hypersensitivity Study in Guinea Pigs, the substance induced and challenged at a 25% concentration produced delayed contact hypersensitivity. When rechallenged at a 10% concentration, the substance did not cause delayed contact hypersensitivity in guinea pigs. 


• In a reliable skin sensitisation test similar to OECD guideline 429, the skin sensitization potential of the read-across analogue substance N-ethylmorpholine was investigated (Holsapple MP, Wiescinsky CM, 1997). Five female Balb/c mice were tested by dose group in four separate trials including 1 vehicle control group and 3 positive control groups. Solutions with 30% vol/vol test substance in acetone/olive oil (4:1) were applied to the ear once daily for three consecutive days. The test item elicited an SI value <3 and is considered to be not skin sensitising.


 


The waivers for in vitro and in vivo testing, considered together with the two available, reliable studies described above on both the test and analogue substance, are considered sufficient to safely conclude on the fact the test substance should not be classified for skin sensitization.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The test substance does not need to be classified as a skin sensitiser according to the criteria laid down in the CLP regulation 1272/2008.