3-(methylthio)propionaldehyde

Acute Toxicity: dermal

Administrative data

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Version / remarks:

adopted 12 May 1981

Deviations:

yes

Remarks:

test item administered as 25% formulation in corn oil, no details on animals and environmental conditions

Principles of method if other than guideline:

An acute peroral toxicity study was conducted in Sprague Dawley rats. The test item was administered by oral gavage as a 25% formulation diluted in corn oil. Each 5 males or 3-5 females received a single oral dose of 2.0, 1.0 and 0.5 mL/kg bw (males) or 4.0, 2.0, 1.0, 0.5 and 0.25 mL/kg bw (females). The animals were inspected daily up to 14 days post dosing for signs of toxic and pharmacological effects. Body weights were recorded prior to administration and 7 and 14 days post-dosing. All animals that died or survived until study termination were necropsied and examined for any signs of gross pathology.

GLP compliance:

not specified

Test type:

standard acute method

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

25% formulation (v/v)

Details on oral exposure:

VEHICLE
- Amount of vehicle: 0.25, 0.5, 1.0, 2.0 and 4.0 mL/kg bw

MAXIMUM DOSE VOLUME APPLIED: 4.0 mL/kg bw

Doses:

0.5, 1.0 and 2.0 mL/kg bw in males, corresponding to 518, 1036 and 2072 mg/kg bw; and 0.25, 0.5, 1.0, 2.0 and 4.0 mL/kg bw in females, corresponding to 259, 518, 1036, 2072 and 4144 mg/kg bw.

No. of animals per sex per dose:

259 mg/kg bw (corresponding to 0.25 mL/kg bw): 3 females
518 mg/kg bw (corresponding to 0.5 mL/kg bw): 5 males and 3 females
1036 mg/kg bw (corresponding to 1.0 mL/kg bw): 5 males and 5 females
2072 mg/kg bw (corresponding to 2.0 mL/kg bw): 5 males and 5 females
4144 mg/kg bw (corresponding to 4.0 mL/kg bw): 5 females

Control animals:

not specified

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations: daily
- Frequency of weighing: before dosing, 7 and 14 days post-dosing
- Necropsy of survivors performed: yes

Key result

Sex:

male

Dose descriptor:

LD50

Effect level:

1 036 mg/kg bw

Based on:

test mat. (dissolved fraction)

95% CL:

611 - 1 761

Remarks on result:

other: corresponding to 1.0 mL/kg bw (95% CL: 0.59 - 1.7 mL/kg bw), dosel levels in mg/kg bw calculated with a density of 1036 mg/mL

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

1 740 mg/kg bw

Based on:

test mat. (dissolved fraction)

95% CL:

984 - 3 098

Remarks on result:

other: corresponding to 1.68 mL/kg bw (95% CL: 0.95 - 2.99 mL/kg bw), dosel levels in mg/kg bw calculated with a density of 1036 mg/mL

Mortality:

259 mg/kg bw (corresponding to 0.25 mL/kg bw): no mortality was observed.
518 mg/kg bw (corresponding to 0.5 mL/kg bw): no mortality was observed.
1036 mg/kg bw (corresponding to 1.0 mL/kg bw): 3/5 males and 1/5 females died within 3.5 - 4 h post-administration.
2072 mg/kg bw (corresponding to 2.0 mL/kg bw): 4/5 males and 3/5 females died within 2 h up to 1 day post-administration.
4144 mg/kg bw (corresponding to 4.0 mL/kg bw): 5/5 females died 1.5 - 4 h post-administration.

Clinical signs:

1036, 2072 and 4144 mg/kg bw (corresponding to 1.0, 2.0 and 4.0 mL/kg bw): sluggishness, salivation, lacrimation, unsteady gait and laboured breathing were seen within 5-15 minutes and prostration within 1 hour post-dosing. Survivors recovered from these effects within 2 days after dosing.

Body weight:

Surviving animals gained body weight as expected.

Gross pathology:

Mottled and red lungs, liquid filled stomachs, red liquid-filled intestines and discolorations of kidneys was noted in all decedent animals. There were no macroscopic findings in surviving animals.

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

The acute oral LD50 was determined to be 1036 mg/kg bw for male rats and 1740 mg/kg bw for female rats.
CLP: Acute Oral 4 (H302) according to Regulation (EC) No. 1272/2008

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

mass median aerodynamic diameter not given

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Principles of method if other than guideline:

- Principle of test: Groups of male and/or female rats were exposed to a saturated vapor atmosphere of the test item for different times of exposure. The test atmosphere was generated statically or dynamically.
- Short description of test conditions:
1. Static exposure: Whole body exposure
120 - 250 g of liquid test substance were placed in an open tray at the top of a sealed 120 or 250 L stainless steel and glass exposure chamber. The atmosphere was allowed to equilibrate for a minimum of 6 h. The animals were then introduced into the chamber through a gasketted door. The test item atmosphere was generated as supplied or from test item which was previously sparged with nitrogen in an attempt to remove volatile impurities, notably acrolein.
The animals were exposed for 24, 40, 49, 60, 80, 97, 100, 195, 240, 360 and 380 min (refer to Table no. 1 under "Any other information on materials and methods incl. tables").
- Parameters analysed / observed: clinical signs, mortality, gross necropsy, histopathological examination of lungs (only from animals exposed for 60 and 240 min); analytical determinations of the test item in the exposure chamber atmosphere.

2. Dynamic exposure: Whole body and nose-only exposure
The atmosphere was generated by metering liquid test substance from a piston pump into a heated evaporator maintained at the lowest temperature (91°C) sufficient to vaporise the liquid.
Whole body exposure was performed in a 900 L stainless steel glass and steel chamber, in which a countercurrent flow of air was passed through the vaporiser and vapor containing air was passed through the chamber.
For nose-only exposure, test item containing air was introduced at the top of the chamber, flowed past the nose-only ports and exhausted at the bottom of the chamber. The atmosphere was sampled at 6 min intervals to measure the test item and acrolein concentration (refer to Table no. 2 under "Any other information on materials and methods incl. tables").
- Parameters analysed / observed: clinical signs, mortality, body weights, gross necropsy, histopathological examination of lungs (only from nose-only exposed animals); analytical determinations of the test item in the exposure chamber atmosphere.

GLP compliance:

not specified

Test type:

traditional method

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

inhalation: vapour

Type of inhalation exposure:

other: whole body and nose only

Vehicle:

air

Remarks:

in some cases sparged with liquid nitrogen, refer to table no. 1

Analytical verification of test atmosphere concentrations:

yes

Remarks:

for 60, 240, 360 and 380 min exposure only

Duration of exposure:

24 - 380 min

Concentrations:

60 min static exposure: 408 ± 4.5, 733 ± 22 and 935 ppm
240 min static exposure: 957 ± 220 ppm
360 min static exposure: 261 ± 20, 270 ± 51 and 328 ± 4.5 ppm
380 min static exposure: 261 ± 2 ppm
(Please refer to Tables no. 1 and 2 under "Any other information on materials and methods incl. tables".)

No. of animals per sex per dose:

5/sex (except for 935 ppm: 3 male, 7 female)

Control animals:

no

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 289 ppm

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: whole body exposure

Remarks:

corresponding to 1.19 ± 0.0081 mg/L air

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 306 ppm

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: nose-only exposure

Remarks:

corresponding to 1.32 ± 0.344 mg/L air

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 320 ppm

Based on:

test mat.

Exp. duration:

1 h

Remarks on result:

other: nose only exposure

Remarks:

corresponding to 1.38 mg/L air

Mortality:

Static exposure (whole body exposure for 24 - 380 min at 261 - 957 ppm): all animal of all concentrations and all exposure durations died (refer to Table no. 3 under "Any other information on results incl. tables").
Dynamic exposure (4 h exposure, whole body at 277 and 289 ppm): no mortality occurred.
Dynamic exposure (1 h exposure, nose-only at 320 ppm): no mortality occurred.
Dynamic exposure (4h exposure, whole body at 306 ppm): 1/5 males and 0/5 females died on Day 4 post exposure.

Clinical signs:

other: Please refer to "Any other information on results incl. tables"

Body weight:

Static exposure (whole body exposure for 24 - 380 min at 261 - 957 ppm): body weights were not recorded
Dynamic exposure (4 h whole body exposure at 277 and 289 ppm): reduced body weight gain during the whole study period
Dynamic exposure (1 and 4 h nose only exposure at 320 and 306 ppm): reduced body weight gain during the first week

Gross pathology:

Static exposure (whole body exposure for 24 - 380 min at 261 - 957 ppm): dark red lungs, clear fluid in the trachea and thoracic cavity, livers with dark purple appearance, epithelium of the trachea and major bronchi were detached and there were areas of eosinophilic fibrillar material along the walls.
Dynamic exposure (4 h whole body exposure at 277 and 289 ppm, 1 and 4 h nose only exposure at 320 and 306 ppm): no abnormalities observed.

Other findings:

Histopathology of lungs:
Static exposure (whole body exposure for 24 - 380 min at 261 - 957 ppm): mild to marked congestion, edema, scattered areas of alvolar haemorrhage.
Dynamic exposure (4 h whole body exposure at 277 and 289 ppm, 1 and 4 h nose only exposure at 320 and 306 ppm): no abnormalities observed.

Clinical signs of toxicity:

Static exposure (whole body exposure for 24 - 380 min at 261 - 957 ppm): lacrimation, nasal discharge, mouth and laboured breathing during exposure, wheezing rales and gasping breathing post-exposure.

Dynamic exposure (4 h whole body exposure at 277 and 289 ppm, 1 and 4 h nose only exposure at 320 and 306 ppm): slight lacrimation, bleopharospasm, occasional slight uniform salivation.

Table 3: Time to death in animals exposed under static conditions

Exposure time [min]	Sex	No. of animals/sex	Atmosphere sparged	Concentration [ppm] mean ± SD	Time of death
24	female	5	No	NM	1-9 days post exposure
40	male	5	No	NM	1-2 days post exposure
49	female	5	No	NM	1-2 days post exposure
60	female	5	No	408 ± 4.5	4 h to 1 day post exposure
60	male/female	3 male, 7 female	No	935	Day 1 post exposure
60	male/female	5	Yes	733 ± 22	Day 1 post exposure
80	male	5	No	NM	1 during exposure, 4 on Day 4 post exposure
97	female	5	No	NM	3 during exposure, 2 on Day 1 post exposure
100	male	5	No	NM	during exposure
195	female	5	No	NM	during exposure
240	male/female	5	No	957 ± 220	during exposure
360	female	5	No	261 ± 20	during exposure
360	female	5	Yes	328 ± 4.5	during exposure
360	male/female	5	Yes	270 ± 51	during exposure
380	male	5	No	261 ± 2.4	during exposure

Interpretation of results:

study cannot be used for classification

Conclusions:

The LD50 values in male and female rats were > 1.19 ± 0.0081 mg/L air (for 4 h whole body exposure) and > 1.32 ± 0.344 mg/L air (for 4 h nose-only exposure). The dose levels were below the recommended limit dose of current guidelines, therefore no conclusion on classification can be drawn.

Cross-reference 3

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

adopted 28 Jul 2015

Deviations:

yes

Remarks:

no erythema and edema values for individual animals reported, only average scores given, occlusive application

GLP compliance:

not specified

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 2.0 - 3.0 kg

Type of coverage:

occlusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.5 mL

Duration of treatment / exposure:

4 h

Observation period:

17 days
Reading time points: 1, 24, 48 and 72 h, and 7, 10, 14 and 17 days

Number of animals:

6 (3 male and 3 female)

Details on study design:

TEST SITE
- Area of exposure: clipped dorsal trunk skin.
- Type of wrap if used: The area was covered with a gauze patch and loosely covered with impervious sheeting.

REMOVAL OF TEST SUBSTANCE
- Occlusive materials were removed and any excess sample wiped away with a moistened gauze swab.
- Time after start of exposure: 4 h

OBSERVATION TIME POINTS : 1, 24, 48 and 72 h, and 7, 10, 14 and 17 days

SCORING SYSTEM:
- Method of calculation: Draize scoring system

Irritation parameter:

erythema score

Basis:

other: average score over all animals

Time point:

24/48/72 h

Score:

2.3

Max. score:

4

Reversibility:

fully reversible within: 17 days

Irritation parameter:

edema score

Basis:

other: average score over all animals

Time point:

24/48/72 h

Score:

2.9

Max. score:

4

Reversibility:

fully reversible within: 17 days

Irritant / corrosive response data:

An hour after removal of the occlusive dressing there was mild to moderate erythema (score 1-2) and well defined to severe edema (score 3-4). On study Days 1-3 mild to severe erythema (score 1-4) was observed, which began to resolve between 3 and 7 days and had disappeared in half of the animals by Day 14 and in all animals by Day 17. Mild to severe edema (score 1-4) was observed in the animals up to study Day 3. One week after patch removal up to study Day 10 slight to moderate edema was observed (score 0-2). 14 days after patch removal, only slight edema (score 0-1) was observed, which was fully reversible until study termination. In addition, desquamation and scab formation were seen in all animals from Day 7-17.

Interpretation of results:

study cannot be used for classification

Conclusions:

The test conditions (occlusive conditions) may have led to more detrimental skin reactions than those specified in current guidelines. Individual animal data for erythema and edema were not reported.

Cross-reference 4

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Remarks:

no details on analytical purity, no details on experimental procedure, no details on animals and environmental conditions, no irritation parameters for individual animals reported, only averaged numbers available

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: Eye irritation in rabbits
- Short description of test conditions: 0.1 mL of the test material were installed into the conjunctival sac of one eye of 6 rabbits (3 male and 3 female, first group) or 0.01 mL were placed on the surface of the cornea of 6 rabbits (3 male and 3 female, second group). The test item was installed as single application without washing. Afterwards, the animals were observed for 21 days.
- Parameters analysed / observed: Eye irritation parameters (corneal opacity, iris, conjunctival redness and chemosis) were scored according to Draize criteria. Average scores over all animals were calculated.

GLP compliance:

not specified

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 2.0 - 3.0 kg

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount applied:
first group: 0.1 mL were instilled into the inferior conjunctival sac of one eye
second group: 0.01 mL were placed on the surface of the cornea

Duration of treatment / exposure:

single application without washing

Observation period (in vivo):

21 days
Reading time points: 1, 24, 48 and 72 h, and 7, 14 and 21 days

Number of animals or in vitro replicates:

0.1 mL test substance instillation into the conjunctival sac of the eye: 6 (3 male and 3 female)
0.01 mL test substance application on the surface of the cornea: 6 (3 male and 3 female)

Details on study design:

SCORING SYSTEM: Draize scoring system

Irritation parameter:

cornea opacity score

Basis:

other: average score over all animals

Time point:

24/48/72 h

Score:

1.5

Max. score:

4

Reversibility:

not fully reversible within: 21 days

Remarks on result:

other: 0.1 mL test substance instillation

Irritation parameter:

iris score

Basis:

other: average score over all animals

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

not fully reversible within: 21 days

Remarks on result:

other: 0.1 mL test substance instillation,

Remarks:

iris could not be scored in some eyes because of corneal opacity

Irritation parameter:

conjunctivae score

Basis:

other: average score over all animals

Time point:

24/48/72 h

Score:

1.4

Max. score:

3

Reversibility:

not fully reversible within: 21 days

Remarks on result:

other: 0.1 mL test substance instillation

Irritation parameter:

chemosis score

Basis:

other: average score over all animals

Time point:

24/48/72 h

Score:

1.83

Max. score:

4

Reversibility:

not fully reversible within: 21 days

Remarks on result:

other: 0.1 mL test substance instillation

Irritation parameter:

cornea opacity score

Basis:

other: average score over all animals

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: 0.01 mL test substance instillation

Irritation parameter:

iris score

Basis:

other: average score over all animals

Time point:

24/48/72 h

Score:

0.37

Max. score:

2

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: 0.01 mL test substance instillation

Irritation parameter:

conjunctivae score

Basis:

other: average score over all animals

Time point:

24/48/72 h

Score:

1.03

Max. score:

3

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: 0.01 mL test substance instillation

Irritation parameter:

chemosis score

Basis:

other: average score over all animals

Time point:

24/48/72 h

Score:

1.1

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: 0.01 mL test substance instillation

Irritant / corrosive response data:

Instillation of 0.1 mL caused marked hyperemia and obvious to severe chemosis, which persisted as such for about 2 days. Chemosis began to resolve by Day 3 and was still present in 2 animals at Day 7 and in mild form in 1 animal at Day 21. Hyperemia, mild to moderate, was still present in all treated eyes by Day 3 and in 5 animals by Day 6. Three animals had residual conjunctival hyperemia at Day 21. Discharge was moderate to marked up to Day 3, resolving in most by Day 14 but still present in 2 animals by Day 21. There was mild corneal injury affecting 25-50% of the cornea at the 1-h reading time point, spreading to the whole of the cornea by the 24-h reading time point, with 1 animal showing opalescent areas at this time. At the 72-h reading time point, 1 animal had opalescent areas, 2 animals had translucent areas, and 3 had just discernible corneal opacity. Most corneal effects cleared in 3 animals by Day 14 and in 4 animals by Day 21. At final inspection on Day 21 one animal had diffuse opalescent changes and another had diffuse opaque cornea with corneal irregularities and neovascularisation.

Application of 0.01 mL test substance to the surface of the cornea caused minor to moderate corneal injury at 4 h post-instillation, which resolved by Day 3. Mild to moderate conjunctival hyperemia, chemosis and discharge was seen 1 h post instillation, which slowly resolved. At the 72-h reading time point, 1 eye had a normal appearance and the remaining 5 animals showed only minor conjunctival irritation.

At 0.1 mL, 2/6 animals developed haemorrhages of the nicitating membrane within 24 hours. 7 days after test item instillation, the cornea one eye had an irregular shape. In 2/6 rabbit eyes there was corneal vascularisation 14 days after test item instillation.

Interpretation of results:

study cannot be used for classification

Conclusions:

Severe and irreversible eye irritating effects were observed in all animals. However, due to limited documentation of effects in individual animals no conclusion on classification is possible.

Cross-reference 5

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 Jul 1997

Deviations:

yes

Remarks:

not tested in TA102 or E. coli WP2 uvrA; 2-aminoanthracene as sole positive control for all strains, no historical control data, no justification for lack of a confirmatory experiment, not tested up to cytotoxic/precipitation limit concentrations

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

The S. typhimurium histidine (his) reversion system measures his- --> his+ reversions. The S. typhimurium strains are constructed to differentiate between base pair (TA 1535, TA 100) and frameshift (TA 1537, TA 98) mutations.

Species / strain / cell type:

other: S. typhimurium TA1535, TA1537, TA1538, TA98 and TA100

Metabolic activation:

with and without

Metabolic activation system:

Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Arochlor 1254.

Test concentrations with justification for top dose:

0.01, 0.0316, 0.1 and 0.316 µg/plate for all strains with and without metabolic activation

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

other: 2-anthramine (2-AM), +S9, 2.5 µg/plate for all strains

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS: triplicates in a single experiment

METHOD OF TREATMENT: pre-incubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h at 37 °C

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Inspection of the bacterial background lawn

METHODS FOR MEASUREMENTS OF GENOTOXICIY :
- Method: Number of revertant colonies

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 0.1 µg/plate with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
A dose-rage finding pre-incubation test was performed with tester strain TA100 with concentrations up to 10 µg/plate in the presence and absence of metabolic activation.
In a pre-incubation assay, four log dilutions of the test substance, to a maximum concentration of 10 µl/plate, were tested. The pre-test was performed with strain TA100 in a concentration range-finding test, with and without metabolic activation. A concentration-related increase in toxicity was observed at 0.1 µg/plate and above.

STUDY RESULTS
- No concentration-related increase in mean histidine revertant colonies/plate was observed for any strain in the absence or presence of metabolic activation.
- Concentration-response relationship: There was no dose-response relationship observed for any test concentration with and without metabolic activation.
- Signs of toxicity: Cytotoxicity was evident by a significant reduction in colonies at 0.316 µl/plate for strain TA1537 in the absence of activation, for all concentrations of test substance strain TA1537 in the presence of activation and for all concentrations of the test substance in strain TA100 in the absence of metabolic activation.
- Concurrent vehicle negative and positive control data: All positive and negative controls gave the expected results and were consistent with those reported in the literature. Historical control data were not reported.
- Mean number of revertant colonies per plate and standard deviation: Refer to Table no. 1 under "Any other information on results incl. tables".

Table 1: Experimental results

Pre-incubation test
Strain	TA 98		TA 100		TA 1535		TA 1537		TA 1538
Metabolic activation	without S9	with S9	without S9	with S9	without S9	with S9	without S9	with S9	without S9	with S9
Vehicle control
DMSO mean	25	41	97	103	14	28	13	29	21	32
± SD	± 4.6	± 6.6	± 12.8	± 7.2	± 3.1	± 12.6	± 4.5	± 6.4	± 7.8	± 5.1
Test item [µg/plate]
0.01 mean	16	31	76	99	19	24	14	15	15	26
± SD	± 4.2	± 4.4	± 5.5	± 5.9	± 2.5	± 2.9	± 3.8	± 4.2	± 5.5	± 7.0
0.0316 mean	19	31	65	97	16	20	13	19	16	28
± SD	± 4.7	± 4.0	± 6.7	± 17.8	± 6.0	± 7.0	± 3.0	± 6.0	± 3.8	± 4.0
0.1 mean	15	31	58	100	22	18	11	17	17	24
± SD	± 4.5	± 4.0	± 6.4	± 5.8	± 5.3	± 5.3	± 3.2	± 0.6	± 1.2	± 5.9
0.316 mean	29	34	65	109	22	20	7	13	13	24
± SD	± 2.3	± 4.0	± 15.0	± 7.0	± 1.2	± 0.6	± 2.6	± 3.8	± 3.5	± 1.5
Positive control
mean	314	1012	1160	2413	1248	479	1256	435	507	1785
± SD	± 10	± 116	± 194	± 352	± 173	± 58	± 89	± 21	± 104	± 269

Conclusions:

Interpretation of results: negative
The test substance did not show mutagenic activity in Salmonella typhimurium TA1535, TA1537, TA1538, TA98 or TA100 with and without metabolic activation.

Cross-reference 6

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted 29 Jul 2016

Deviations:

yes

Remarks:

limited information on experimental details and raw data of individual replicates

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

mouse lymphoma thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: L5178Y mouse lymphoma cells, clone 3.7.2c

For cell lines:
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: The cells were cultivated in Fisher's medium supplemented with 31 µg penicillin/mL, 50 µg streptomycin sulfate/mL, 0.1% pluronic F68, 0.22 mg sodium pyruvate/mL, 25 mM HEPES buffer. In the absence of metabolic activation 10% heat-inactivated horse serum were added, in the absence of metabolic activation 5% heat-inactivated horse serum were added.

Metabolic activation:

with and without

Metabolic activation system:

Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Arochlor 1254.

Test concentrations with justification for top dose:

Experiment 1:
Without S9 mix: 0.0001, 0.01, 0.0178, 0.0316 and 0.0562 µL/mL (4 h)
With S9 mix: 0.0562, 0.1, 0.178, 0.316 and 0.562 µL/mL (4 h)

Experiment 2:
Without S9 mix: 0.001, 0.01, 0.015, 0.02, 0.025, 0.03, 0.035 and 0.04 µL/mL (4 h)
With S9 mix: 0.01, 0.05, 0.10, 0.15, 0.20 and 0.25 µL/mL (4 h)

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: hycanthone (-S9), 7.5 and 10.0 µg/mL

Details on test system and experimental conditions:

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 h with and without S9 mix

FOR GENE MUTATION:
- Expression time: 48 h
- Selection time: 14 days
- Fixation time: 16 days
- Selection agent: 1 µg/mL trifluorothymidine (TFT)
- Method to enumerate numbers of viable and mutants cells: relative total growth

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Relative cloning efficiency, relative total growth

METHODS FOR MEASUREMENTS OF GENOTOXICIY :
- Number and size distribution of small and large colonies and total number of colonies
- Mutant frequency

Evaluation criteria:

Colonies in the mutant count and cloning efficiency dishes were counted, and the colonies in the solvent and positive control mutant count plates were sized by hand using a dissecting microscope to enumerate mutant colonies.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

0.02 µL/mL, 0.15 µL/mL

Vehicle controls validity:

valid

Remarks:

cloning efficiency was below acceptability criteria in the first test; vehicle controls were valid in the second test

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: In the main mutagenicity study no precipitation was observed, neither in the presence nor in the absence of metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
In the range-finding assay, both in the presence and absence of metabolic activation, there were concentration-related decreases in cell survival. No cells were present in cultures exposed to >0.1 µL/mL in the absence of metabolic activation or in cultures exposed to >1.0 µL/mL with metabolic activation. Slight precipitate was seen without activation at >0.316 µL/mL and with metabolic activation at >0.1 pl/ml. Based on these findings, the first mutagenesis assay was conducted using a concentration range of 0.0001-0.0562 µL/mL without metabolic activating and 0.0562-0.562 µL/mLl with metabolic activation.

STUDY RESULTS:
In the first mouse lymphoma assay, there was a concentration-dependent increase in mutant frequencies, as well as a concentration-dependent increase in induced mutant frequencies (please refer to Table 1 under “Any other information on results incl. tables”). However, no concentration was tested that yielded survival in the 10-20% range. In addition, the average cloning efficiencies of the solvent controls were 62.7% without S9 mix and 55.4% with S9 mix, which were below the acceptability criteria of 70%. Mutant frequencies obtained with the solvent control were high, especially in the presence of metabolic activation, suggesting the preparation may have been mutagenic. For this reason, the mutagenicity assay was repeated.
In the second assay, average cloning efficiencies of solvent controls and spontaneous mutant frequencies were within the expected range. In addition, there was a dose-related decrease in relative total growth and a concentration-related increase in mutant frequency, with and without metabolic activation (please refer to Table 2 under “Any other information on results incl. tables”). The test item induced clear concentration-related increases in induced colonies up to cytotoxic concentrations (relative total growth values ≥ 10%). Further, the test item induced clear increases in small colonies (< 1 mm in diameter) and only relatively slight increases in large colonies, both with and without metabolic activation.
In both experiments, positive controls showed the expected results and were within the range of historical control data (data not provided in publication).


ADDITIONAL INFORMATION ON CYTOTOXICITY:
In a first assay with concentrations of 0.0001 to 0.0562 µL/mL  without metabolic activation and 0.0562 to 0.562 µL/mL with metabolic activation no concentration tested yielded survival in the range of 10 to 20%. Aaverage cloning efficiencies of the solvent controls were below the acceptability criteria. Therefore a second assay with concentrations of 0.001 to 0.04 µL/mL without S9  and 0.01 to 0.25 µL/mL with S9 was conducted. The cloning efficiencies of the solvent control in the second test were considered acceptable.

Table 1: Experimental results, first mouse lymphoma assay

Test group	RSG (%)	RTG (%)	MF x 10^6	IMF x 10^6
Without metabolic activation, 4 h exposure
DMSO control
10	88.8	90.9	81	-
10	111.2	108.6	82	-
Test item [µL]
0.0001	88.1	97.7	92	11
0.0010	75.7	98.4	87	6
0.0100	71.4	88.8	63	-
0.0178	52.1	62.7	107	26
0.0316	33.7	37.6	116	35
0.0562	0.8	-	-	-
HC 7.5 µg	26.9	14.5	685	604
HC 10.0 µg	23.5	12.1	690	609
With metabolic activation, 4 h exposure
DMSO control
10	112.3	104.0	291	-
10	87.7	94.2	363	-
Test item [µL]
0.05620	86.0	94.7	361	34
0.10000	80.5	91.3	364	37
0.17800	76.2	72.9	381	54
0.31600	52.9	55.7	356	29
0.56200	14.9	6.7	596	269
CP 2.5 µg	50.2	19.2	862	535
CP 3.75 µg	47.2	2.7	977	650

HC: Hycanthone, CP: Cyclophosphamide, RSG: relative suspension growth, RTG: relative total growth, MF: mutant frequency, IMF: induced mutant frequency

Table 2: Experimental results, second mouse lymphoma assay

Test group	RSG (%)	RTG (%)	MF x 10^6	IMF x 10^6	Colony formation MF x 10^6
					small	large	total
Without metabolic activation, 4 h exposure
DMSO control
10	102.0	105.2	70	-	31	34	70
10	98.0	94.9	58	-	28	30	58
Test item [µL]
0.0010	89.7	101.0	56	-	24	39	56
0.0100	81.0	106.5	77	13	43	34	77
0.0150	61.0	71.0	94	30	69	25	94
0.0200	57.9	47.7	117	53	86	30	117
0.0250	45.0	35.2	144	80	118	26	144
0.0300	28.0	17.4	174	110	123	51	174
0.0350	17.7	8.0	281	217	217	64	281
0.0400	7.7	2.4	545	482	427	115	545
HC 7.5 µg	46.1	33.0	296	233	203	93	296
HC 10.0 µg	50.4	27.0	289	225	211	78	289
With metabolic activation, 4 h exposure
DMSO control
10	88.2	97.2	43	-	22	20	43
10	111.8	100.4	47	-	22	24	47
Test item [µL]
0.010	99.9	91.4	41	-	15	26	41
0.050	89.2	81.9	26	-	16	10	26
0.100	68.4	48.3	67	23	52	15	67
0.150	45.9	27.0	85	40	59	26	85
0.200	23.5	11.5	228	184	194	35	228
0.250	10.2	2.4	647	602	538	109	647
CP 2.5 µg	97.7	40.2	221	176	177	44	221
CP 3.75 µg	61.1	14.5	317	272	269	48	317

HC: Hycanthone, CP: Cyclophosphamide, RSG: relative suspension growth, RTG: relative total growth, MF: mutant frequency, IMF: induced mutant frequency

Conclusions:

Interpretation of results: positive with and without metabolic activation.

Cross-reference 7

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

adopted 17 Jul 1992

Deviations:

yes

Remarks:

no challenge control, limited information on materials and methods

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Covance, Inc., Denver, Pennsylvania, USA
- Age at study initiation: 5-7 weeks
- Housing: individually in suspended, stainless steel cages with wire mesh bottoms
- Diet: Certified Guinea Pig Diet No. 5026 (PMI Nutrition International, St. Louis, Missouri), ad libitum
- Water: tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16-25
- Humidity (%): 20-62
- Photoperiod (hrs dark / hrs light): 12/12

Route:

intradermal and epicutaneous

Vehicle:

propylene glycol

Concentration / amount:

intradermal induction on Day 1: 5% in propylene glycol
epicutaneous induction on Day 7: 0.2 mL unchanged

Day(s)/duration:

intradermal induction: single treatment on Day 0; epicutaneous induction: 24 h exposure on Day 6

Adequacy of induction:

other: concentration chosen based on the results of a preliminary range-finding study in which 100% produced a score of 0.5 in 2/6 animals

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

0.1 mL

Day(s)/duration:

Day 21, 48 h

Adequacy of challenge:

other: concentration chosen based on the results of a preliminary range-finding study in which 100% produced a score of 0.5 in 2/6 animals

No.:

#2

Route:

epicutaneous, occlusive

Vehicle:

propylene glycol

Concentration / amount:

25%

Day(s)/duration:

Day 21, 48 h

Adequacy of challenge:

other: The concentration was chosen due to excess irritation in the control group after the first challenge.

No. of animals per dose:

20 (10 male and 10 female)

Details on study design:

RANGE FINDING TESTS:
Induction and maximum non irritant concentration for challenge was determined in a pre-test: 5% MTPA in propylene glycol administered intradermally produced local necrosis. 25% topical application produced no irritation, and up to 100% only produced a score of 0.5 in 2 of 6 animals.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal and epicutaneous, respectively)
- Exposure period: single injection (intradermal) and 24 h (epicutaneous)
Intradermal (3 pairs of injections):
Injection 1: 50% complete Freund's adjuvant (FCA) in distilled water
Injection 2: 5% test substance in propylene glycol
Injection 3: 5% test substance in 50% aqueous FCA
Epicutaneous: 7 days after the injections, 0.2 mL of undiluted test substance were applied on a 2 x 4 inch patch of filter paper and applied to the reclipped areas which had received the intradermal injections. The patches were covered by impermable plastic and secured with an elastic adhesive bandage wound around the animals torso. After 48 h occluded contact the skin was wiped free of excess test material with gauze soaked in 0.9% saline.
- Control group: Not included in the study.


B. CHALLENGE EXPOSURE
- No. of exposures: 2
- Day of challenge: 2 weeks after epidermal induction (Day 20)
- Exposure period: 24 h
- Test groups: test substance
- Control group: Not included in the study.
- Concentrations: 0.1 mL undiluted test item
- Evaluation (hr after challenge): 24 and 48 h

Challenge controls:

No challenge control group was included.

Positive control substance(s):

yes

Remarks:

a-hexylcinnamaldehyde

Positive control results:

Two positive control groups were included. Both groups were induced intradermally with 50% FCA in distilled water, 30% a-hexylcinnamaldehyde (HCA) in propylene glycol, and 30% HCA in 50% aqueous FCA. Epicutaneous induction was with a 48 h occluded contact with undiluted HCA. Animals were challenged with both 50% HCA in propylene glycol and undiluted HCA. Challenge with undiluted HCA induced a skin sensitisation of a score of 1 or greater in 40% of the animals. The severity indices were 0.6 (24 h) and 0.35 (48 h). Challenge with 50% HCA induced a positive response in 90% of the animals with severity indices of 1.1 (24 h) and 0.9 (48 h).

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

induction: 5% intradermal and 0.2 mL unchanged epicutaneous induction, challenge with 100% epicutaneous induction

No. with + reactions:

3

Total no. in group:

19

Remarks on result:

other: 1/10 females died on Day 10

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

induction: 5% intradermal and 0.2 mL unchanged epicutaneous induction, challenge with 100% epicutaneous induction

No. with + reactions:

5

Total no. in group:

19

Remarks on result:

other: 1/10 females died on Day 10

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

induction with vehicle, untreated during challenge

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: 4/10 animals gave a result of score 1. In view of this, a score of 2 or greater was used as basis for a positive response.

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

induction: 30% intradermal and 0.2 mL unchanged epicutaneous induction, challenge with 100% epicutaneous induction

No. with + reactions:

1

Total no. in group:

10

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

induction: 30% intradermal and 0.2 mL unchanged epicutaneous induction, challenge with 100% epicutaneous induction

No. with + reactions:

0

Total no. in group:

10

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

induction: 5% intradermal and 0.2 mL unchanged epicutaneous induction, challenge with 25% epicutaneous induction

No. with + reactions:

2

Total no. in group:

19

Remarks on result:

other: re-challenge, 1st reading

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

induction: 5% intradermal and 0.2 mL unchanged epicutaneous induction, challenge with 25% epicutaneous induction

No. with + reactions:

4

Total no. in group:

19

Remarks on result:

other: re-challenge, 2nd reading

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

induction: 30% intradermal and 0.2 mL unchanged epicutaneous induction, challenge with 50% epicutaneous induction

No. with + reactions:

2

Total no. in group:

10

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

induction: 30% intradermal and 0.2 mL unchanged epicutaneous induction, challenge with 50% epicutaneous induction

No. with + reactions:

0

Total no. in group:

10

Results of pilot study:
Intradermal injections of 5% MTPA produced local
necrosis and the concentration was considered adequate for
the main study. Topical application of 25% produced no
local irritation and 50, 70, 100% produced a score of
0.5 in 2 or 3 of 6 animals. Therefore undiluted MTPA was used for the
main study epicutaneous induction and challenge.

Table 1: Results of main study - Incidences of skin responses at challenge and in irritation control animals

Compound	Group	No. of animals	Time	Erythema Score					Edema	Necrosis
				0	0.5	1	2	3
Test item, 100%	Challenge	19	24	14	1	1	0	3	5	3
		19	48	8	5	1	0	5	11	4
Test item, 25%	Re-challenge	19	24	12	4	0	0	2	3	0
		19	48	3	8	3	0	4	13	2
Test item, 100%	Irritation	10	24	8	0	0	0	2	2	2
		10	48	6	2	0	0	0	4	1
Test item, 25%	Irritation	10	24	5	5	0	0	0	3	0
		10	48	1	5	4	0	0	6	0
HCA, 100%	Challenge	10	24	2	4	3	1	0	2	0
		10	48	5	3	2	0	0	0	0
HCA, 100%	Irritation	10	24	8	2	0	0	0	0	0
		10	48	10	0	0	0	0	0	0
HCA, 50%	Challenge	10	24	0	2	6	2	0	1	0
		10	48	0	2	8	0	0	0	0
HCA, 50%	Irritation	10	24	4	5	1	0	0	0	0
		10	48	6	4	0	0	0	0	0

HCA: a-hexylcinnamaldehyde
One female of the MTPA sensitization group died on day 10 of the study.
The irritation reaction was unexpectedly high compared to the  pre-study.

In summary, based on the scores of 2 or greater with epicutaneous challenge at 25% the test item  produced sensitization in 22% of the guinea pigs; therefore,this material was classified as a "mild sensitizer" based on the Magnusson and 

Kligman allergenicity rating criteria.

Interpretation of results:

study cannot be used for classification

Conclusions:

Based on the experimental findings, the test item causes erythema, edema and necrosis to the skin. However, due to the highly skin irritating properties, a distinct conclusion on skin sensitising effects cannot be drawn. Therefore the skin sensitisation study test result is considered to be inconclusive due to irritating effects of the test item to the skin.

Cross-reference 8

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Version / remarks:

adopted 12 May 1981

Deviations:

yes

Remarks:

limited study duration including 9 days treatment instead of 21/28 days, treatment 5 days/week instead of daily application, test material not fully characterised

GLP compliance:

yes

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 165.3 - 195.5 g (males) and 182.5 - 216.0 g (females)
- Housing: individually in stainless steel wire mesh cages
- Diet: Certified Rodent Diet No. 5000 (PMI Nutrition International, St. Louis, Missouri), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 26
- Humidity (%): 24 - 70

Type of coverage:

occlusive

Vehicle:

unchanged (no vehicle)

Details on exposure:

TEST SITE
- Area of exposure: The doses were applied to the clipped dorsal trunk skin.
- Type of wrap if used: The treated areas were covered with a layer of 8-ply gauze and impervious plastic, which was secured with an adhesive elastic bandage.

REMOVAL OF TEST SUBSTANCE
- Washing: The test sites were wiped free of excess test material using gauze soaked in saline.
- Time after start of exposure: 6 h

TEST MATERIAL
- Amount applied: 0.05, 0.2 and 0.5 mL/kg bw/day, corresponding to 52.7, 210.8 and 527 mg/kg bw/day
- Constant volume or concentration used: no

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

6 h

Frequency of treatment:

6 hours/day for 9 days (5 days in first week, 4 days in second week)

Dose / conc.:

0.05 other: mL/kg bw

Remarks:

corresponding to 52.7 mg/kg bw/day

Dose / conc.:

0.2 other: mL/kg bw

Remarks:

corresponding to 210.8 mg/kg bw/day

Dose / conc.:

0.5 other: mL/kg bw

Remarks:

corresponding to 527 mg/kg bw/day

No. of animals per sex per dose:

Main group: 10
Recovery group: 5 (control and high dose group only)

Control animals:

yes

Details on study design:

- Dose selection rationale: Dose levels were chosen based on a preliminary range-finding study, which was performed in 3 rats/sex with treatment for 5 consecutive days. 1/3 males and 1/3 females treated at 1.0 mL/kg bw/day died on study Day 4. There were no clinical signs of toxicity and no abnormalities in the Irwin Screen. Local skin irritation, seen at 0.5 and 1.0 mL/kg bw/day, was mainly mild erythema. Body weight losses at 0.5 mL/kg bw/day and 1.0 mL/ kg bw/day were significantly greater than for the control group. Except for the 1.0 mL/kg bw/day males, all groups gained weight by the fifth day of dosing. Based on these findings, it was considered that applied doses of 0.05, 0.2, and 0.5 mL/kg bw/day were appropriate for the full 9-day study.
- Fasting period before blood sampling for clinical biochemistry: not specified
- Post-exposure recovery period in satellite groups: 28 days

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily during the exposure period, once weekly during the recovery period
- Cage side observations included: local skin lesions and signs of toxic or pharmacological effects and mortality

DERMAL IRRITATION: Yes
- Time schedule for examinations: daily during the exposure period, once weekly during the recovery period

BODY WEIGHT: Yes
- Time schedule for examinations: on study Days 1, 3, 5, 8 and 10 during the exposure period and on Day 15 and weekly thereafter during the recovery period.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Food consumption was measured for a week prior to dosing, over Days 1-3, 3-5, 5-8 and 8-10 during the exposure period and for recovery animals over six daily intervals.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: Prior to dosing, over Days 1-3, 3-5, 5-8 and 8-10 during the exposure period and for recovery animals over six daily intervals.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was collected before sacrifice from the retro-orbital venous plexus and used for chemical pathological measurements.
- Anaesthetic used for blood collection: no data
- Animals fasted: no data
- How many animals: all animals
- Parameters checked: hemoglobin concentration, hematocrit, erythrocyte count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet and reticulocyte counts, total and differential leukocyte counts, prothrombin time, and activated partial thromboplastin time.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was collected before sacrifice from the retro-orbital venous plexus and used for chemical pathological measurements.
- Anaesthetic used for blood collection: no data
- Animals fasted: no data
- How many animals: all animals
- Parameters checked: urea nitrogen, glucose, total protein, albumin, globulin, bilirubin, sodium, potassium, calcium, chloride, inorganic phosphorus, aspartate and alanine aminotransferases, alkaline phosphate, lactate and sorbitol dehydrogenases, creatine kinase, and y-glutamyl transferase.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected over a 2 h interval from trays under the animal’s cage, and over 16 h from animals in metabolism cages.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters checked: Appearance, microscopy, pH, protein, ketones, bilirubin, occult blood, urobilinogen, glucose, creatinine, specific gravity, osmolality, and N-acetyl-ß-D-glucosaminidase (NAG) activity.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Before dosing, after the fifth dosing and before necropsy. Recovery animals were also evaluated the day following the cessation of dosing and prior to sacrifice.
- Dose groups that were examined: all groups
- Type of test: Irwing Screen, functional observation battery

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. Necroscopic examination was conducted on all animals sacrificed by CO2 inhalation at the ends of the dosing and recovery periods to detect any indications of gross pathological change. Complete macroscopic postmortem examinations were conducted on all animals.
- The following organs were removed for weighing: brain, adrenal glands, liver, kidneys, ovaries, and testes.

HISTOPATHOLOGY: Yes. Histopathological evaluation of selected tissues (brain, kidneys, nerves, skin, spinal cord and testes) were performed on controls and high dose animals. Tissues and organs were removed, fixed in 10% neutral-buffered formalin, and then processed for light microscopic examination.

Clinical signs:

no effects observed

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

Local signs of irritation were barely perceptible erythema in a few of the low-dose animals. Most mid- and high-dose animals showed very slight to slight erythema. By Day 18, all high-dose recovery animals were free of skin irritation.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gains of the high-dose males and females were lower than for the controls over the treatment period. At the end of the dosing period (Day 12) high-dose males had mean body weights 7% lower than controls, and high-dose females 5% lower. Body weights for the mid- and low-dose groups were comparable to the controls. Gains during the recovery period were comparable for control and high-dose animals.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

The mean food consumption per bw. in all treated groups of males was 10 to 15% (p<0.01) higher than in controls from Day 5 until Day 10 but not dose-related. The effect was considered to be non-adverse.

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment related histopathologic findings in the skin of the high-dose animals consisted of an occasional trace to mild inflammatory cell infiltrates in the dermis. The effect was considered adverse.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Mild to moderate hyperkeratosis and acanthosis were observed in all animals of all dose groups at the treated skin site and acute inflammatory cell infiltration was noted in high dose-groups animals only. The effects were considered treatment-related and toxicologically relevant.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

527 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect observed

Remarks on result:

other: corresponding to 0.5 mL/kg bw/day

Key result

Dose descriptor:

LOAEL

Remarks:

local

Effect level:

527 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

dermal irritation

histopathology: non-neoplastic

Remarks on result:

other: corresponding to 0.5 mL/kg bw/day

Key result

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

210.8 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect observed

Remarks on result:

other: corresponding to 0.2 mL/kg bw/day

Key result

Critical effects observed:

no

Conclusions:

Application of the test material to the skin of Sprague Dawley rats for a period of 9 days produced mild local skin reactions at 0.2 mL/kg bw/day (corresponding to 210.8 mg/kg bw/day) and mild to moderate local skin reactions at 0.5 mL/kg bw/day (corresponding to 527 mg/kg bw/day). The findings of the high dose group persisted until study Day 17. Based on these findings, a LOAEL for local toxicity of 527 mg/kg bw/day was derived and a NOAEL for local toxicity of 210.8 mg/kg bw/day. The NOAEL for systemic toxicity was 0.5 mL/kg bw/day (corresponding to 527 mg/kg bw/day).

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Cutaneous toxicology of 3-(Methylthio)propionaldehyde.

Author:

Ballantyne B., Cawley T.J., Blaszcak D.L.

Year:

2000

Bibliographic source:

J.Toxicol.-Cut. & Ocular Toxicol., 19 (2&3), 117-130

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1986

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Version / remarks:

adopted 09 Oct 2017

Deviations:

yes

Remarks:

occlusive dressing, no stepwise dosing

GLP compliance:

not specified

Test type:

standard acute method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3-(methylthio)propionaldehyde

EC Number:

221-882-5

EC Name:

3-(methylthio)propionaldehyde

Cas Number:

3268-49-3

Molecular formula:

C4H8OS

IUPAC Name:

3-(methylthio)propionaldehyde

Test animals

Species:

rabbit

Strain:

New Zealand White

Sex:

male/female

Administration / exposure

Type of coverage:

occlusive

Vehicle:

unchanged (no vehicle)

Details on dermal exposure:

TEST SITE
- Area of exposure: clipped dorsal trunk
- Type of wrap if used: The test material was applied to the test site and the exposed skin was covered with gauze and impervious sheeting. The materials were held in place using an elastic bandage around the trunk.

Duration of exposure:

24 h

Doses:

0.25, 0.5, 1.0 mL/kg bw (males), corresponding to 259, 518 and 1036 mg/kg bw and 0.5, 1.0 mL/kg bw (females), corresponding to 518 and 1036 mg/kg bw

No. of animals per sex per dose:

4

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations: during exposure and daily thereafter
- Frequency of weighing: before dosing, at 7 and 14 days post dosing
- Necropsy of survivors performed: yes

Results and discussion

Effect levels

Effect levels 1

Sex:

male

Dose descriptor:

LD50

Effect level:

736 mg/kg bw

Based on:

test mat.

95% CL:

446 - 1 191

Remarks on result:

other: dosel levels in mg/kg bw calculated with a density of 1036 mg/mL

Effect levels 2

Sex:

female

Dose descriptor:

LD50

Effect level:

818 mg/kg bw

Based on:

test mat.

95% CL:

508 - 1 347

Remarks on result:

other: dosel levels in mg/kg bw calculated with a density of 1036 mg/mL

Mortality:

0.25 mL/kg bw (corresponding to 259 mg/kg bw): 1/4 males died on Day 9.
0.5 mL/kg bw (corresponding to 518 mg/kg bw): no mortality occurred.
1.0 mL/kg bw (corresponding to 1036 mg/kg bw): 4/4 males and 3/4 females died (males: death within 3 h - 5 days; females: death within 1 day).

Clinical signs:

Local signs: Marked erythema, edema and necrosis in all animals of all dose groups, which persisted 14 days. Desquamation and fissuring were seen at 7 days in females at 1 mL/kg bw (1036 mg/kg bw).
Clinical signs: Salivation, sluggishness, and unsteady gait. Survivors recovered from these effects by 2 days.

Body weight:

Body weight gain was unaffected by treatment.

Gross pathology:

The main necropsy finding was light to dark red mottling of the lungs. Sacrificed survivors had no gross pathological changes observed.

Applicant's summary and conclusion

Interpretation of results:

Category 3 based on GHS criteria

Conclusions:

CLP: Acute Dermal 3, H311 according to Regulation (EC) No. 1272/2008