Dimethyl(propyl)amine

Endpoint summary

Description of key information

The results of the studies were as follows:

The test item was considered to be irritating to rabbits' skin according to OECD 404 and corrosive in the in vitro study according to OECD 431. Since in vivo data are preferred to in vitro data, the in vivo test with rabbits was retained as key study for assessment of the skin irritating potential of the test item.

The test item DMPA causes severe eye damage to rabbits' eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

07 June - 21 June 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

- Lot/batch No.: K322/1

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain as cited in the study report: White New Zealand/Chbb:NZW (SPF)
- Source: Boehringer Ingelheim Pharma KG
- Age at study initiation: young adult animals
- Weight at study initiation: 4, 2.8, 2.9 kg
- Housing: single housing in stainless steel wire mesh cages with grating, floor area 3000 cm2
- Diet: Kliba-Labordiet (Klingenthalmuehle AG, Kaiseraugst, Switzerland), about 130 g per animal and day
- Water: 250 mL tap water per animal and day
- Acclimation period: at least 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark/hrs light): 12/12

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Remarks:

(24 hrs prior to testing)

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

Amount applied: 0.5 mL

Duration of treatment / exposure:

4 hours

Observation period:

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

Number of animals:

3 male animals

Details on study design:

TEST SITE
- Area of exposure: 2.5 cm x 2.5 cm
- Type of wrap used: test patch (Idealbinde, Pfaelzische Verbandstoff-Fabrik, Kaiserslautern) and stretch (Fixomull adhesive fleece, Baiersdorf)

REMOVAL OF TEST SUBSTANCE
- Washing: after removal of the dressing, the application site was washed with Lutrol followed by Lutrol in water (1:1).
- Time after start of exposure: 4 hours

SCORING SYSTEM: Scoring was done as specified by the OECD guideline, according to the Draize scoring system.

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

3

Max. score:

4

Reversibility:

not fully reversible within: 14 days

Remarks on result:

other: erythema grade 2 still was seen after 14 days.

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2.7

Max. score:

4

Reversibility:

not fully reversible within: 14 days

Remarks on result:

other: erythema grade 1 still was seen after 14 days.

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: erythema grade 1 was seen at each of reading time points 1, 24, 48 and 72 hrs, with full reversibility reached at day 7.

Irritation parameter:

erythema score

Basis:

mean

Remarks:

out of all 3 animals

Time point:

24/48/72 h

Score:

2.2

Max. score:

4

Reversibility:

not fully reversible within: 14 days

Remarks on result:

other: at day 14, 2/3 animals still showed erythema grade 1 and 2, respectively.

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

not fully reversible within: 14 days

Remarks on result:

other: edema grade 1 still was seen at day 14.

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.7

Max. score:

4

Reversibility:

fully reversible within: 14 days

Remarks on result:

other: edema grade 1 was seen at reading time points 48 h, 72 h, and at day 7 where it was accompanied by scaling. The finding was no more evident at day 14.

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: reversibility: not applicable

Remarks on result:

other: this animal showed no edema at all.

Irritation parameter:

edema score

Basis:

mean

Remarks:

out of all 3 animals

Time point:

24/48/72 h

Score:

0.6

Max. score:

4

Reversibility:

not fully reversible within: 14 days

Remarks on result:

other: 1/3 animals still showed edema grade 1 accompanied by scaling at day 14.

Irritant / corrosive response data:

Slight (1/3 animals) to moderate (2/3 animals) erythema was seen after 1 h. At 24 h, erythema became severe in one case and remained moderate and slight in the 2 other animals, respectively. At 48 and 72 h, 2/3 animals has severe erythema whereas the third animal still showed a slight effect. After 7 days, the two cases of severe erythema turned back to moderate whereas full reversibility was noticed for the third animal. After 14 days, one animal still showed moderate erythema whereas in the other one, the effect turned back to slight.
Regarding edema, whereas no effect was seen at reading time point 1 h, slight edema appeared in 1/3 animals at 24 h, and in 2/3 animals at 48 h. The effect in the 2 animals lasted until day 7 and was further accompanied by scaling. At day 14, only 1 of these 2 animals still showed edema with scaling.

Other effects:

scaling seen at day 7 (2/3 animals) and at day 14 (1/3).

Table 1: Details on findings and scores:

	Animal	Erythema  score	Edema  score
1h	1	2	0
	2	2	0
	3	1	0
24 hrs	1	3	1
	2	2	0
	3	1	0
48 hrs	1	3	1
	2	3	1
	3	1	0
72 hrs	1	3	1
	2	3	1
	3	1	0
7 d	1	2	1 + severe scaling
	2	2	1 + scaling
	3	0	0
14 d	1	2	1 + scaling
	2	1	0
	3	0	0

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

DMPA was tested for skin irritation in rabbits, under semi-occlusive conditions, according to the OECD TG 404 (1992). After 4 h exposure, the animals were observed during 14 days, with the skin being examined for signs of irritation at the following reading time points: 1, 24, 48 and 72 h, day 7 and day 14. The skin findings were assessed by scoring according to the Draize scoring system.
All 3 animals showed slight to severe erythema from 1 to 72 h; at day 7 and 14 slight to moderate erythema still was noticed in 2/3 animals, indicating that full reversibility was not reached at the end of the observation period of 14 days.
Slight edema appeared in 1/3 animals after 24 h and was seen in 2/3 animals at 48 h, 72 h and at day 7. At day 14, 1/3 animal still displayed slight edema. At 7 and 14 day, edema was accompanied by scaling.
Taking into account the mean erythema and edema scores obtained from all 3 animals over the reading time points 24, 48 and 72 h, and regarding the absence of full reversibility at day 14, the test item is to be considered as a skin irritant according to current evaluation criteria.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

29 May 2012 - 14 June 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

- Lot/batch No.: 11-09-LR15

Test system:

human skin model

Source species:

human

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Episkin TM Model Kit (0.38 cm2 tissues) supplied by SkinEthic Laboratories, Nice, France.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (or 37°C (?) - clarification required)
- Temperature of post-treatment incubation: 3 hours +/- 5 minutes

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 12 times with 2 mL D-PBS

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 minutes, 1 hour and 4 hours
- Spectrophotometer:
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: duplicates

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 35%, or if the viability after 3 minutes exposure is greater than or equal to 35 % and the viability after 1 hour exposure is less than 35% OR if viability is greater than or equal to 35% after 1 hours and less than 35% after 4 hours.
- The test substance is considered to be non-corrosive to skin if the viability after 4 hour exposure is greater than or equal to 35%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 50 µL

Duration of treatment / exposure:

3 minutes, 1 hour, and 4 hours.

Number of replicates:

duplicate

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min exposure

Value:

94

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min exposure

Value:

27

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

4 hours exposure

Value:

15

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

other: Category 1B and 1C based on GHS

Executive summary:

The objective of this study was to evaluate the corrosive potential of the test item, using the EpiskinTM reconstituted skin model. The study was conducted according to the OECD TG 431. Duplicate tissues were treated with the test item for each of following time-exposure: 3 minutes, 1 hour, and 4 hours. DMPA was tested as such, i.e., undiluted. As a test substance may directly reduce MTT, a preliminary assay was performed adding 50 µL test item to 2.2 mL of a 0.3 mg/mL freshly prepared MTT solution. The mixture was incubated in darkness for 3 hours. A negative control was tested concurrently. As the MTT solution colour did not turn blue/purple when compared to the negative control, the dosage form was presumed not to reduce MTT. As a result, no additional controls were performed on water-killed tissues in parallel to the main test (performed on viable tissues). In the main test, the test item, negative and positive controls were applied on duplicate tissues and incubated at room temperature as follows: positive and negative controls for 4 hours (± 5 minutes); test item for 3 minutes (± 5 seconds), 1 hour (± 5 minutes) and 4 hours (± 5 minutes). At the end of the designated incubation period, each tissue insert was rinsed and transferred into two empty wells of each 12-well plate containing MTT. Each plate was protected from light and incubated for 3 hours (± 5 minutes) at room temperature. At the end of the MTT incubation period, the tissues were removed from its plastic insert. Any tissue discolouration was evaluated with the naked eye (discoloured surface area and intensity). Then, for each tissue, the epidermis was separated from the collagen matrix and both parts were put into acidified isopropanol overnight to extract the formazan (reduced MTT) out of the MTT-loaded tissues. At the end of the extraction period, the optical density of each extract was measured at 570 nm. The 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).

The relative mean viabilities of the test item-treated tissues were 94% for the 3 minute exposure, 27% for the 1 hour exposure and 15% for the 4 hours exposure. The blue discoloration of the test item-treated tissues following the 3-minute and 1-hour exposure periods was representative of viable tissue. The blue/white discolouration of the test item-treated tissues following the 4-hour exposure period was representative of semi-viable tissue. The acceptance criteria of the test were fulfilled. Based on the results of the study, DMPA is considered to be corrosive to the skin when tested in the in vitro skin corrosion test according to OECD TG 431.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

28 June 1999- 05 July 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

1987

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

- Lot/batch No.: K322/1

Species:

rabbit

Strain:

Himalayan

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Strain as cited in the study report: Himalayan/Chbb:HM (outbred strain)
- Source: Boehringer Ingelheim Pharma KG
- Age at study initiation: young adult animals
- Weight at study initiation: 2.4 kg
- Housing: single housing in stainless steel wire mesh cages with grating, floor area 3000 cm2
- Diet: Kliba-Labordiet (Klingenthalmuehle AG, Kaiseraugst, Switzerland), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark/hrs light): 12/12

Vehicle:

unchanged (no vehicle)

Controls:

other: the untreated eye served for control

Amount / concentration applied:

Amount applied: 0.1 mL

Duration of treatment / exposure:

24 h

Observation period (in vivo):

7 days
reading time points: 1, 24, 48 and 72 h, and day 7

Number of animals or in vitro replicates:

1 animal (female)

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: with tap water
- Time after start of exposure: 24 hours

SCORING SYSTEM: scoring of the findings was done as recommended by the OECD guideline, according to the Draize scoring system.

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2.3

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: corneal opacity grade 3 still was seen at the end of the observation period, i.e. at day 7.

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

not fully reversible within: 72 hrs

Remarks on result:

other: alteration of the iris grade 1 was seen at 24, 48 and 72 h; due to severe lesions of the eye on day 7, the iris could not be assessed at that time point.

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

3

Max. score:

3

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: conjunctival redness grade 3 still was seen at the end of observation period of 7 days.

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2.3

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Remarks on result:

other: chemosis grade 3 still was seen at the end of observation period of 7 days.

Irritant / corrosive response data:

The animal displayed corneal opacity, conjunctival redness and chemosis as well as discharge during to whole period of observation. Regarding the iris, a reaction grade 1 was seen at 24, 48 and 72 h; because of additional alterations, the iris could not be evaluated at day 7.
A discharge grade of 3 was seen, i.e. with moistening of the lids and hairs affecting a considerable area around the eye, still was seen at the end of observation period of 7 days.

Other effects:

The additional alterations almost consisted of suppuration, bloody discharge, loss of corneal tissue, and discolouration of parts of the conjunctiva and nictitating membrane (yellow-brown to white). These alterations were seen all over the reading time points, from 1 h to day 7.

Table 1: Details on findings and scores:

Readings	animal	cornea score	iris score	conjunctiva redness	chemosis	discharge	additional alterations
1 hr	1	2	0	2	2	2	bloody discharge, loss of corneal tissue, parts of conjunctiva and nictitating membrane yellow-brown discoloured (indicating necrosis in the view of the study director)
24 hrs	1	2	1	3	2	3	bloody discharge, loss of corneal tissue, parts of conjunctiva and nictitating membrane yellow-brown discoloured (indicating necrosis in the view of the study director)
48 hrs	1	2	1	3	2	3	bloody discharge, parts of conjunctiva and nictitating membrane yellow-brown/white discoloured (indicating necrosis in the view of the study director)
72 hrs	1	3	1	3	3	3	suppuration, bloody discharge, parts of conjunctiva and nictitating membrane yellow-brown/white discoloured (indicating necrosis in the view of the study director)
7 d	1	3		3	3	3	suppuration, bloody discharge, loss of corneal tissue, parts of conjunctiva and nictitating membrane white discoloured (indicating necrosis in the view of the study director). Due to the severity of the findings, the study was stopped.
mean		2.3	1	3	2.3

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Taking into account the mean scores for corneal opacity, iris, conjunctiva redness and chemosis over the reading time points 24, 48 and 72 h, and regarding the severity of the additional lesions and the absence of reversibility, the test item is to be considered as causing serious damages to the eye according to current evaluation criteria.

Executive summary:

DMPA was tested for eye irritation in one female rabbit according to the OECD TG 405 (1987). The test item was applied undiluted into the conjunctival sac of one eye. After 24 h exposure, the eye was washed and examined for signs of irritation at the following reading time points: 1, 24, 48 and 72 h, and day 7. Due to the severity of the findings, the test was stopped at day 7. The eye findings were assessed by scoring according to the Draize scoring system.

Severe occular lesions affecting the cornea, iris, conjunctiva and the lids were detected at all observation time points; thus, the effects were not reversible within the 7 day period of observation. Additional lesions also were reported, including severe discharge, suppuration, discharge with blood, and yellow-brown to white discolouration of the conjunctiva and nictitating membrane, which, according to the study director, indicated necrosis. No further animals needed to be tested.

Taking into account the mean scores for corneal opacity, iris, conjunctiva redness and chemosis over the reading time points 24, 48 and 72 h, and regarding the severity of the additional lesions and the absence of reversibility, the test item is to be considered as causing serious damages to the eye according to current evaluation criteria.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

Skin irritation/corrosion

In vivo study

DMPA was tested for skin irritation in rabbits, under semi-occlusive conditions, according to the OECD TG 404 (2000). After 4 h exposure, the animals were observed during 14 days, with the skin being examined for signs of irritation at the following reading time points: 1, 24, 48 and 72 h, day 7 and day 14. The skin findings were assessed by scoring according to the Draize scoring system. All 3 animals showed slight to severe erythema from 1 to 72 h; at day 7 and 14 slight to moderate erythema still was noticed in 2/3 animals, indicating that full reversibility was not reached at the end of the observation period of 14 days. Slight edema appeared in 1/3 animals after 24 h and was seen in 2/3 animals at 48 h, 72 h and at day 7. At day 14, 1/3 animal still displayed slight edema. At 7 and 14 day, edema was accompanied by scaling.

Taking into account the mean erythema and edema scores obtained from all 3 animals over the readings time point 24, 48 and 72 h, and regarding the absence of full reversibility at day 14, the test item is to be considered as a skin irritant according to current evaluation criteria.

In vitro study

An in vitro corrosion test according to the OECD TG 431 was conducted with DMPA to evaluate the corrosive potential of the test item, using the EpiskinTM reconstituted skin model (CIT 39105) (2012). Duplicate tissues were treated with the undiluted test item for 3 minutes, 1 hour, and 4 hours. As a test item may directly reduce MTT, a preliminary assay was performed. The MTT solution colour did not turn blue/purple when compared to the negative control, thus, the dosage form was presumed not to reduce MTT and no additional controls were performed on water-killed tissues in parallel to the main test (performed on viable tissues). In the main test, the test item, negative and positive controls were applied on duplicate tissues and incubated at room temperature as follows: positive and negative controls for 4 hours (± 5 minutes); test item for 3 minutes (± 5 seconds), 1 hour (± 5 minutes) and 4 hours (± 5 minutes).Thereafter, each tissue was incubated with MTT. Any tissue discolouration was first evaluated visually and then each tissue was subjected to formazan extraction (i.e. reduced MTT), and the optical density of each extract was measured at 570 nm.

The relative mean viabilities of the test item-treated tissues were 94% for the 3 minute exposure, 27% for the 1 hour exposure and 15% for the 4 hours exposure. The blue discoloration of the test item-treated tissues following the 3-minute and 1-hour exposure periods was representative of viable tissue. The blue/white discolouration of the test item-treated tissues following the 4-hour exposure period was representative of semi-viable tissue. The acceptance criteria of the test were fulfilled. Based on the results of the study, DMPA is considered to be corrosive to the skin when tested in the in vitro skin corrosion test according to OECD TG 431.

The differences might at least in part be explained by the very high vapour pressure of the substance, which limits skin contact in vivo, but evaporation is more limited in the in vitro settings. Because of the more realistic situation in the rabbit study and because in vivo data are generally preferred to in vitro data, the in vivo test with rabbits was retained as key study for assessment of the skin irritating potential of the test item.

Eye irritation/corrosion

DMPA was tested for eye irritation in one female rabbit according to the OECD TG 405 (2000). The test item was applied undiluted into the conjunctival sac of one eye. After 24 h exposure, the eye was washed and examined for signs of irritation at the following reading time points: 1, 24, 48 and 72 h, and day 7. Due to the severity of the findings, the test was stopped at day 7. The eye findings were assessed by scoring according to the Draize scoring system.

Severe occular lesions affecting the cornea, iris, conjunctiva and the lids were detected at all observation time points; thus, the effects were not reversible within the 7 day period of observation. Additional lesions also were reported, including severe discharge, suppuration, discharge with blood, and yellow-brown to white discolouration of the conjunctiva and nictitating membrane, which, according to the study director, indicated necrosis. No further animals needed to be tested.

Taking into account the mean scores for corneal opacity, iris, conjunctiva redness and chemosis over the reading time points 24, 48 and 72 h, and regarding the severity of the additional lesions and the absence of reversibility, the test item is to be considered as causing serious damages to the eye according to current evaluation criteria.

Respiratory irritation

Based on the clinical symptoms observed and described within the acute inhalation study (2012) entered in chapter 7.2.2, respiratory irritation is expected.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No 1272/2008. As a result the substance is considered to be classified as skin irritant Cat. 2 (H 315) and as causing irreversible eye damage Cat. 1 (H 318) under Regulation (EC) No 1272/2008 (CLP). Furthermore, the test item is classified as STOT SE Cat. 3 and labelled with H335 under Regulation (EC) No 1272/2008.