Registration Dossier

Administrative data

Description of key information

According to the negative results of the in vitro skin irritation test (OECD 439) and no erythema observed in the acute dermal toxicity study (OECD 402), DPTH is not classified as irritating to the skin.
Negative result is showed in the in vivo irritation test (OECD 405) ; therefore DPTH is not classified as irritating to the eyes.
No irritation on respiratory tract was observed in the acute toxicity study by inhalation.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 August 2011 to 31 October 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline No. 439
Deviations:
yes
Remarks:
see below
Qualifier:
according to guideline
Guideline:
other: Commission Regulation (EC) No. 761/2009, B.46
Deviations:
yes
Remarks:
see below
Principles of method if other than guideline:
The study was performed in accordance with study plan No. 38014 TIC with the following deviations from the agreed study plan:
. during the treatment of the tissues with the positive control (SDS 5%), the tissue No. 3 was treated with a volume < 10 µL. As the acceptance criteria for the positive controls were fulfilled (viability of the tissue No. 3 is < 40% and the SD value between the tissue replicates viabilities are < 18%), this deviation was considered to not have compromised the validity or integrity of the study,
. during the test for direct MTT reduction, the test item was incubated with a freshly prepared MTT solution for 3 hours and 7 minutes instead of 3 hours ± 5 minutes. This deviation was considered to not have compromised the validity or integrity of the study.
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
other: reconstituted human epidermis
Cell source:
other: reconstituted human epidermis
Details on animal used as source of test system:
Episkin TM Model Kit (0.38 cm2 tissues) supplied by SkinEthic Laboratories, Lyon, France.
Medium and Incubation T°C: 37°C
Dates of experimental phase: from 30 August 2011 to 31 October 2011.
Vehicle:
unchanged (no vehicle)
Control samples:
yes, concurrent no treatment
yes, concurrent positive control
Amount/concentration applied:
As the test item was a solid, 5 µL of water for injection was first applied to each of the three tissues and then, 10 mg ± 2 mg of the test item were applied evenly to each tissue, taking care to spread it over the whole tissue surface without damaging the tissue sample.
Duration of treatment / exposure:
The test item, the negative and positive controls were applied topically on triplicate tissues and incubated at room temperature during 15 (± 1) minutes. At the end of the treatment period, each tissue was rinsed with D-PBS and incubated for 42 (± 1) hours at 37°C, 5% CO2 in a humidified incubator.
Duration of post-treatment incubation (if applicable):
At the end of the 42-hour incubation period, the cell viability was then assessed by means of the colorimetric MTT reduction assay.
Number of replicates:
Tripicate tissues for each tested substance (test item, negative control, positive control)
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
treated
Value:
100.5
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Preliminary test
In the preliminary test, thetest item was found to not have direct MTT reducing properties since the MTTsolution containing the test item did notturn blue/purple when compared to the negative control.As a result, no additional controls were performed on water-killed tissues in parallel to the main test.
The test item was found to not have a coloring potential in the preliminary test since the water solution containing the test item did not change color. As a result, no additional controls were used in parallel to the main test.

Main test
All acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered as valid.
Following a 15-minute exposure and a 42-hour recovery period, the relative mean viability of the tissues treated with the test item was 100.5%.
 Group Tissue No.  OD measurements (first) OD measurements (second) Mean OD blank  cOD first   cOD second Mean cOD   Viability (%)
 Negative control  1  1.033 1.004   0.996  0.967  0.982  96.0 
   2  1.089 1.054   0.037 1.052  1.017  1.035  101.2 
   3  1.108 1.108    1.071  1.032  1.052  102.8 
 Positive control  1  0.131 0.129    0.094  0.092  0.093  9.1 
   2  0.095 0.089  0.037  0.058 0.052  0.055  5.4 
   3  0.294 0.300    0.257  0.263  0.260  25.4 
 Test item  1  1.093 1.080    1.056  1.043 1.050  102.6 
   2  1.042 1.026  0.037  1.005  0.989 0.997  97.5 
   3  1.087  1.058   1.050  1.021  1.036  101.3 

OD = optical density

cOD = blanck corrected optical density

Interpretation of results:
GHS criteria not met
Remarks:
not skin irritating
Conclusions:
The test item is considered as non-irritant to skin in this in vitro test.
Executive summary:

The objective of this study was to evaluate the skin irritation potential of the test item,Dipentamethylenethiuram hexasulfide,using the EpiskinTM reconstituted human epidermis model.

The study design is based on international guidelines (OECD Guideline No. 439 and Commission Regulation (EC) No. 761/2009, B.46) and thestudy was conducted in compliance with CIT’s standard operating procedures and the principles of Good Laboratory Practice.

Methods

Preliminary tests were performed to detect the ability of the test item to directly reduce MMT as well as its coloring potential.

Following the preliminary tests, the skin irritation potential of the test item was tested in one main test. The test item, the negative and positive controls were applied topically on triplicate tissues and incubated at room temperature during 15 (± 1) minutes. At the end of the treatment period, each tissue was rinsed with D-PBS and incubated for 42 (± 1) hours at 5% CO2 in a humidified incubator. The cell viability was then assessed by means of the colorimetric MTT reduction assay.

Relative viability values were calculated for each tissue and expressed as percentages of the negative control tissues viability which was set at 100% (reference viability).

 

Results

Preliminary test

In the preliminary test, thetest item was found to not have direct MTT reducing properties since the MTTsolution containing the test item did notturn blue/purple when compared to the negative control.As a result, no additional controls were performed on water-killed tissues in parallel to the main test.

The test item was found to not have a coloring potential in the preliminary test since the water solution containing the test item did not change color. As a result, no additional controls were used in parallel to the main test.

Main test

All acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered as valid.

Following a 15-minute exposure and a 42-hour recovery period, the relative mean viability of the tissues treated with the test item was 100.5%.

 

Conclusion

The test item is considered as non-irritant to skin.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 October 2011 - 21 November 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
yes
Remarks:
- the relative humidity recorded in the animal room was sometimes outside of the target ranges specified in the study plan,no pH measurement was performed as the test item was insoluble in water.
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
yes
Remarks:
idem above
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Breeder: Hypharm, La Corbière, Roussay, France
- Age at study initiation: at the beginning of the study, the animals were 2 to 4 months old
- Mean body weight at study initiation: the animals had a mean body weight ± standard deviation of 3.323 kg ± 0.129 kg
- Fasting period before study: no
- Housing: individually housed in noryl cages
- Diet: free access to pelleted breeding diet “type 110C”
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: at least 5 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 ± 3°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h (7:00 - 19:00).

IN-LIFE DATES: 09 November 2011 to 21 November 2011
Vehicle:
unchanged (no vehicle)
Controls:
other: untreated right eye served as a control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.1 g/animal.
Duration of treatment / exposure:
Not applicable: single application not followed by rinsing.
Observation period (in vivo):
1, 24, 48 and 72 h; if relevant, daily until reversibility of reactions
Number of animals or in vitro replicates:
3 males.
Details on study design:
REMOVAL OF TEST SUBSTANCE: No

SCORING SYSTEM: Draize scale.

- Conjunctival chemosis (lids and/or nictitating membranes):
0 no swelling
1 any swelling above normal (includes nictitating membranes)
2 obvious swelling with partial eversion of lids
3 swelling with lids about half-closed
4 swelling with lids more than half-closed

- Conjunctival redness (palpebral and bulbar conjunctivae, cornea and iris):
0 blood vessels normal
1 a number of blood vessels definitely hyperemic (injected)
2 diffuse, crimson colour, individual vessels not easily discernible
3 diffuse, beefy red

- Discharge:
0 absence of discharge
1 slight discharge (does not include small amounts normally found in inner canthus)
2 discharge with moistening of lids and hairs adjacent to lids
3 discharge with moistening of lids and hairs on wide area around the eye

- Iris lesions
0 normal
1 markedly deepened rugae, congestion, swelling, moderate circum-corneal hyperemia,or injection, any of these or combination of any thereof, iris still reacting to light (sluggish reaction is positive)
2 no reaction to light, haemorrhage, gross destruction (any or all of these)

- Cornea intensity of opacity (direct examination and, if necessary, with an UV lamp)
0 no ulceration or opacity
1 scattered or diffuse areas of opacity (other than slight dulling or normal lustre), details of iris clearly visible
2 easily discernible translucent area, details of iris slightly obscured
3 nacreous areas, no details of iris visible, size of pupil barely discernible
4 opaque cornea, iris not discernible through the opacity

- Cornea area of opacity (direct examination and, if necessary, with an UV lamp)
1 one quarter (or less) but not zero
2 greater than one quarter but less than a half
3 greater than one half but less than three quarters
4 greater than three quarters up to whole area

- Any other lesions observed were noted

TOOL USED TO ASSESS SCORE: UV lamp after instillation of 0.5% sodium fluorescein solution
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
3 rabbits
Time point:
24/48/72 h
Score:
0.53
Max. score:
4
Reversibility:
fully reversible within: 6 days
Remarks on result:
other: Individual scores : 0.3 -1.3 -0.0 showing no significant eye irritation
Irritation parameter:
conjunctivae score
Remarks:
(redness)
Basis:
mean
Remarks:
3 rabbits
Time point:
24/48/72 h
Score:
0.77
Max. score:
3
Reversibility:
fully reversible within: 72 hours
Remarks on result:
other: Individual scores: 0.7 -1.3 -0.3 showing no significant eye irritation.
Irritation parameter:
iris score
Basis:
mean
Remarks:
3 rabbits
Time point:
24/48/72 h
Score:
0.23
Max. score:
2
Reversibility:
fully reversible within: 72 hours
Remarks on result:
other: Individual scores: 0.0 -0.7-0.0 showing no significant eye irritation
Irritation parameter:
cornea opacity score
Remarks:
(opacity)
Basis:
mean
Remarks:
3 rabbits
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 72 hours
Remarks on result:
other: Individual scores: 0.0 -1.0 -0.0 showing eye irritation in 1/3 animals.
Irritant / corrosive response data:
First animal (No. 57): A moderate chemosis (grade 2) was observed at the 1-hour observation, became slight 24 hours after treatment and had disappeared on day 3. A severe redness of the conjunctiva (grade 3) was observed 1 hour after treatment, then became slight on days 2 and 3, and had disappeared on day 4.
No iris or corneal lesions were observed.

Second animal (No. 58): A moderate chemosis (grade 2) was noted on days 1 and 2 and was then graded slight from day 3 to day 5. A severe redness of the conjunctiva (grade 3) was observed on days 1 and 2, was slight on day 3 and was no longer observed from day 4. Iris lesions (grade 1) were noted on days 2 and 3. A slight corneal opacity (intensity: grade 2 to 1 and area: grade 1) was recorded on days 2 and 3 and had completely recovered on day 4.
A whitish purulent discharge was also observed on days 2 to 4.

Third animal (No. 59): A moderate chemosis (grade 2) was observed at the 1-hour observation only, together with a severe redness of the conjunctiva (grade 3). The redness became slight on day 2 and was no longer observed from day 3. No iris or corneal lesions were observed.
Other effects:
No unscheduled deaths occurred during the study.
No clinical signs other than ocular reactions were noted in any animals.
The body weight of the animals was unaffected by the test item-treatment.

First animal

 

 

 

 

 

 

Irritation parameter

Basis

Time point

Score

Max. score

Reversibility

Remarks

chemosis

mean

24, 48 and 72 h

0.3

1

fully reversible within: 48h

 

conj

mean

24, 48 and 72 h

0.7

1

fully reversible within: 72h

 

iris

mean

24, 48 and 72 h

0

0

other: not applicable

 

cornea

mean

24, 48 and 72 h

0

0

other: not applicable

 

 

 

 

 

 

 

 

 second animal

 

 

 

 

 

 

Irritation parameter

Basis

Time point

Score

Max. score

Reversibility

Remarks

chemosis

mean

24, 48 and 72 h

1.3

2

fully reversible within: 6 days

 

conj

mean

24, 48 and 72 h

1.3

3

fully reversible within: 72h

 

iris

mean

24, 48 and 72 h

0.7

1

fully reversible within: 72h

 

cornea

mean

24, 48 and 72 h

1

2

fully reversible within: 72h

 

 

 

 

 

 

 

 

Third animal

 

 

 

 

 

 

Irritation parameter

Basis

Time point

Score

Max. score

Reversibility

Remarks

chemosis

mean

24, 48 and 72 h

0

0

other: not applicable

 

conj

mean

24, 48 and 72 h

3

1

fully reversible within: 48h

 

iris

mean

24, 48 and 72 h

0

0

other: not applicable

 

cornea

mean

24, 48 and 72 h

0

0

other: not applicable

 

 

 

 

 

 

 

 

Interpretation of results:
GHS criteria not met
Remarks:
not eye irritating
Conclusions:
The test item was slightly irritant when administered by the ocular route to rabbits.
Executive summary:

The objective of this study was to evaluate the potential eye irritant properties of the test item, Dipentamethylenethiuram hexasulfide, following a single administration to rabbits.

This study was conducted in compliance with OECD guideline (OECD 404) and with the principles of Good Laboratory Practice.

Methods

The test item Dipentamethylenethiuram hexasulfidewas first administered to a single male New Zealand White rabbit.

As mean value from grading at 24, 48 and 72 hours after instillation was < 2 for conjunctival edema (chemosis) or for conjunctival redness and/or < 1 for iris lesion or for corneal opacity, the test item was administered in the left eye of two other animals.

 

The test item was administered inthe conjunctival sac of the left eye. The right eye remained untreated and served as control.A quantity of 0.1 g/animal was used. Just after the 1-hour scoring, both eyes were rinsed with a sterile isotonic saline solution (0.9% NaCl).

 

Each animal was observed at least once a day for mortality and clinical signs.Ocular reactions were observed approximately 1 hour, 24, 48 and 72 hours after the administration and then daily until the reversibility of the ocular reactions (daytwo animals, daythe other one). The mean values of the scores for chemosis, redness of the conjunctiva, iris lesions and corneal opacity were calculated for each animal. Body weight was recorded on the day of treatment and the end of the observation period.

On completion of the observation period, the animals were sacrificed then discarded without macroscopic post-mortem examination.

Results

Moderate chemosis and severe redness of the conjunctiva were observed in all animals on day 1 and/or 2. Then slight conjunctival reactions persisted up to day 5 at the latest.

Iris lesions and a moderate then slight corneal opacity were noted in 1/3 animals on days 2 andaddition, whitish purulent discharge was observed from day 2 until day 4.

 

Mean scores calculated for each animal over 24, 48 and 72 hours were as follows:

.       chemosis: 0.3, 1.3 and 0.0; showing no significant eye irritation,

.       redness of the conjunctiva: 0.7, 1.3 and 0.3; showing no significant eye irritation,

.       iris lesions: 0.0, 0.7 and 0.0; showing no significant eye irritation,

.       corneal opacity: 0.0, 1.0 and 0.0; showing eye irritation in 1/3 animals.

 

No ocular reactions were observed in the right eye of all animals.

 

Conclusion

The test item was slightly irritant when administered by the ocular route to rabbits.

However, the test item is not classified as irritating to the eyes according to the criteria of CLP Regulation.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

In vitro skin irritation test (OECD 439):

The objective of this study was to evaluate the skin irritation potential of the test item, Dipentamethylenethiuram hexasulfide, using the Episkin reconstituted human epidermis model (OECD Guideline No. 439 and Commission Regulation (EC) No. 761/2009, B.46).

Preliminary tests were performed to detect the ability of the test item to directly reduce MMT as well as its coloring potential.

In the preliminary test, the test item was found to not have direct MTT reducing properties since the MTT solution containing the test item did not turn blue/purple when compared to the negative control. As a result, no additional controls were performed on water-killed tissues in parallel to the main test. The test item was found to not have a coloring potential in the preliminary test since the water solution containing the test item did not change color. As a result, no additional controls were used in parallel to the main test.

In the main test, all acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered as valid.

Following a 15-minute exposure and a 42-hour recovery period, the relative mean viability of the tissues treated with the test item was 100.5%.

The test item is considered as non-irritant to skin.

In vivo eye irritation test (OECD 405):

The objective of this study was to evaluate the potential eye irritant properties of the test item, Dipentamethylenethiuram hexasulfide, following a single administration to rabbits (OECD guideline no. 404).

The test item Dipentamethylenethiuram hexasulfide was administered in the conjunctival sac of the left eye to three male New Zealand White rabbits. The right eye remained untreated and served as control.A quantity of 0.1 g/animal was used. Just after the 1-hour scoring, both eyes were rinsed with a sterile isotonic saline solution (0.9% NaCl).

Each animal was observed at least once a day for mortality and clinical signs.Ocular reactions were observed approximately 1 hour, 24, 48 and 72 hours after the administration and then daily until the reversibility of the ocular reactions.

No clinical signs other than ocular reactions were noted in any animals. The body weight of the animals was unaffected by the test item-treatment.

Moderate chemosis and severe redness of the conjunctiva were observed in all animals on day 1 and/or 2. Then slight conjunctival reactions persisted up to day 5 at the latest. Iris lesions and a moderate then slight corneal opacity were noted in 1/3 animals on days 2 andaddition, whitish purulent discharge was observed from day 2 until day 4.

Mean scores calculated for each animal over 24, 48 and 72 hours were as follows: chemosis: 0.3, 1.3 and 0.0; showing no significant eye irritation, redness of the conjunctiva: 0.7, 1.3 and 0.3; showing no significant eye irritation, iris lesions: 0.0, 0.7 and 0.0; showing no significant eye irritation, corneal opacity: 0.0, 1.0 and 0.0; showing eye irritation in 1/3 animals. 

No ocular reactions were observed in the right eye of all animals.

Under the experimental conditions of this study, the test item, Dipentamethylenethiuram hexasulfide, was slightly irritant when administered by the ocular route to rabbits.

 

Justification for classification or non-classification

Based on the available data, no classification for irritation is required for Dipentamethylenethiuram hexasulfide according to the Regulation EC n°1272/2008.