Registration Dossier

Administrative data

Endpoint:
acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April -September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Qualifier:
according to
Guideline:
EU Method B.3 (Acute Toxicity (Dermal))
GLP compliance:
yes (incl. certificate)
Test type:
standard acute method
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: bulk
Details on test material:
- Name of test material (as cited in study report): Diacid 1550, K-salt (trade name: "H-240 100%")
- Physical state: amber solid
- Storage condition of test material: At room temperature in original, sealed container
Specific details on test material used for the study:
Identification: H-240 100% a.s.
Synonyms: Diacid H-240; OCD 9142-055
CAS Number: 68127-33-3
Purity: 100%
Physical state/Appearance: amber colored solid
Expiry Date: 10 April 2018
Storage Conditions: room temperature in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Five male and five female Wistar (RccHanTm:WIST) strain rats were supplied by Envigo RMS (UK) Limited, Oxon, UK. On receipt the animals were randomly allocated to cages. The females were nulliparous and non-pregnant. After an acclimatization period of at least 5 days the animals were selected at random and given a number unique within the study by indelible ink-marking on the tail and a number written on a cage card. At the start of the study the animals weighed at least 200 g, and were 8 to 12 weeks of age. The weight variation did not exceed ±20% of the mean weight for each sex.
The animals were housed in suspended solid floor polypropylene cages furnished with woodflakes. The initial two animals were housed individually throughout the study. The further group of eight animals (four male and four female) were housed individually during the 24-hour exposure period and in groups of four, by sex, for the remainder of the study. Free access to mains drinking water and food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study.
The diet, drinking water and bedding were routinely analyzed and were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
The temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70%, respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Administration / exposure

Type of coverage:
semiocclusive
Vehicle:
other: For the purpose of the study the test item was weighed out according to each animal's individual body weight and moistened with distilled water prior to application.
Duration of exposure:
24 h
Doses:
2000 mg/kg
No. of animals per sex per dose:
5 males (2000 mg/kg), 5 females (2000 mg/kg)
Control animals:
no
Details on study design:
On the day before treatment the back and flanks of each animal were clipped free of hair.
In the absence of data suggesting the test item was toxic, one male and one female rat were initially treated with the test item at a dose level of 2000 mg/kg.
The appropriate amount of test item, moistened with distilled water, was applied as evenly as possible to an area of shorn skin (approximately 10% of the total body surface area). A piece of surgical gauze was placed over the treatment area and semi-occluded with a piece of self-adhesive bandage. The animals were caged individually throughout the study. Shortly after dosing the dressings were examined to ensure that they were securely in place.
After the 24-hour contact period the bandage was carefully removed and the treated skin and surrounding hair wiped with cotton wool moistened with distilled water to remove any residual test item.
As no mortalities were noted a further group of animals (four males and four females) was similarly treated with the test item at a dose level of 2000 mg/kg body weight to give a total of five males and five females. The animals were caged individually for the 24-hour exposure period. After the 24-hour contact period the bandages were carefully removed and the treated skin and surrounding hair wiped with cotton wool moistened with distilled water to remove any residual test item. These animals were returned to group housing for the remainder of the test period.
The animals were observed for deaths or overt signs of toxicity 30 minutes, 1, 2 and 4 hours after dosing and subsequently once daily for 14 days.
After removal of the dressings and subsequently once daily for 14 days, the test sites were examined for evidence of primary irritation and scored according to the following scale:

EVALUATION OF SKIN REACTIONS

Erythema and Eschar Formation
Value
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beef redness) to slight eschar formation (injuries in depth) 4

Edema Formation
No edema 0
Very slight edema (barely perceptible) 1
Slight edema (edges of area well-defined by definite raising) 2
Moderate edema (raised approximately 1 millimeter) 3
Severe edema (raised more than 1 millimeter and extending beyond the area of exposure) 4

Any other skin reactions, if present were also recorded.
Individual body weights were recorded prior to application of the test item on Day 0 and on Days 7 and 14.
At the end of the study the animals were killed by cervical dislocation. All animals were subjected to gross necropsy. This consisted of an external examination and opening of the abdominal and thoracic cavities. The appearance of any macroscopic abnormalities was recorded. No tissues were retained.
Statistics:
The following computerized system was used in the study:
Delta Controls — ORCAview

Results and discussion

Preliminary study:
Initially, two animals (one male and one female) were given a single, 24 hour, semi-occluded dermal application of the test item to intact skin at a dose level of 2000 mg/kg body weight. As no mortalities were noted a further group of animals (four males and four females) was similarly treated with the test item at a dose level of 2000 mg/kg body weight to give a total of five males and five females.
Effect levels
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
There were no deaths
Clinical signs:
No signs of systemic toxicity were noted during the observation period
Body weight:
One female showed no gain in body weight during the first week with expected gain in body weight during the second week and one other female showed expected gain in body weight during the first week but body weight loss during the second week. The remaining animals showed expected gains in body weight over the study period.
Gross pathology:
No abnormalities were noted at necropsy
Other findings:
Dermal reactions: Very slight or well-defined erythema and very slight or slight edema were noted at the test sites of all animals. Hemorrhage of dermal capillaries and/or small superficial scattered scabs were noted at the test sites of three animals. Scab cracking was also noted at the test site of one male. Loss of skin elasticity and flexibility was noted at the test sites of two males.

Any other information on results incl. tables

Individual clinical observations and mortality data

Dose
Level
mg/lig

Animal
Number
and Sex

Effects Noted After Dosing
(Hours)

Effects Noted During Period After Dosing

(Days)

Y2

 1

2

4

1

2

3

4

5

6

7

8

9

10

11

12

13

14

2000

1-0
Male

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

3-0
Male

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

3-1
Male

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

3-2
Male

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

3-3
Male

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

2-0
Female

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

4-0
Female

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

4-1
Female

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

4-2
Female

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

4-3
Female

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0 = No signs of systemic toxicity

Individual Dermal Reactions - Males

Dose Level mg/kg

Animal
Number
and Sex

Observation

Effects Noted After Initiation of Exposure (Days)

1

2

3

4

5

6

7

8

9

10

11

12

13

14

2000

1-0
Male

Erythema

2

1

1

0

0

0

0

0

0

0

0

0

0

0

Edema

2

1

0

0

0

0

0

0

0

0

0

0

0

0

Other

0

0

0

0

0

0

0

0

0

0

0

0

0

0

3-0
Male

Erythema

2

2

2

0

0

0

0

0

0

0

0

0

0

0

Edema

2

2

2

0

0

0

0

0

0

0

0

0

0

0

Other

0

Hd

Ss

Ss

0

0

0

0

0

0

0

0

0

0

3-1
Male

Erythema

2

1

0

0

0

0

0

0

0

0

0

0

0

0

Edema

2

1

0

0

0

0

0

0

0

0

0

0

0

0

Other

0

0

LeLf

0

0

0

0

0

0

0

0

0

0

0

3-2
Male

Erythema

2

2

2

0

0

0

0

0

0

0

0

0

0

0

Edema

2

2

2

0

0

0

0

0

0

0

0

0

0

0

Other

0

0

Ss

SsSk

Ss

Ss

Ss

Ss

Ss

0

0

0

0

0

3-3
Male

Erythema

2

1

0

0

0

0

0

0

0

0

0

0

0

0

Edema

2

1

0

0

0

0

0

0

0

0

0

0

0

0

Other

0

0

LeLf

0

0

0

0

0

0

0

0

0

0

0

0 = No reactions

Hd = Hemorrhage of dermal capillaries

Ss = Small superficial scattered scabs         

0 = Due to a technician error observation not recorded

Individual Dermal Reactions - Females

Dose Level mg/kg

Animal
Number
and Sex

Observation

Effects Noted After Initiation of Exposure (Days)

1

2

3

4

5

6

7

8

9

10

11

12

13

14

2000

2-0
Female

Erythema

1

0

0

0

0

0

0

0

0

0

0

0

0

0

Edema

1

0

0

0

0

0

0

0

0

0

0

0

0

0

Other

0

0

0

0

0

0

0

0

0

0

0

0

0

0

4-0
Female

Erythema

2

2

1

0

0

0

0

0

0

0

0

0

0

0

Edema

2

2

1

0

0

0

0

0

0

0

0

0

0

0

Other

0

Hd

Ss

Ss

Ss

Ss

Ss

Ss

Ss

0

0

0

0

0

4-1
Female

Erythema

2

1

1

0

0

0

0

0

0

0

0

0

0

0

Edema

2

1

0

0

0

0

0

0

0

0

0

0

0

0

Other

0

0

0

0

0

0

0

0

0

0

0

0

0

0

4-2
Female

Erythema

2

1

0

0

0

0

0

0

0

0

0

0

0

0

Edema

1

1

0

0

0

0

0

0

0

0

0

0

0

0

Other

0

0

0

0

0

0

0

0

0

0

0

0

0

0

4-3
Female

Erythema

2

2

1

0

0

0

0

0

0

0

0

0

0

0

Edema

2

1

0

0

0

0

0

0

0

0

0

0

0

0

Other

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0 = No reactions

Hd = Hemorrhage of dermal capillaries

Ss = Small superficial scattered scabs         

0 = Due to a technician error observation not recorded

Individual Body Weights and Body Weight Changes

Dose Level
mg/kg

Animal Number
and Sex

Body Weight (g) at Day

Body Weight Change (g) During Week

0

7

14

1

2

2000

1-0 Male

260

282

319

22

37

3-0 Male

287

306

326

19

20

3-1 Male

281

303

320

22

17

3-2 Male

284

305

328

21

23

3-3 Male

269

284

302

15

18

2-0 Female

222

250

245

28

—5

4-0 Female

234

235

247

1

12

4-1 Female

228

228

238

0

10

4-2 Female

230

241

257

11

16

4-3 Female

225

230

241

5

11

Individual Necropsy Findings

Dose Level
mg/kg

Animal Number
and Sex

Time of Death

Macroscopic Observations

2000

1-0
Male

Killed Day 14

No abnormalities detected

3-0
Male

Killed Day 14

No abnormalities detected

3-1
Male

Killed Day 14

No abnormalities detected

3-2
Male

Killed Day 14

No abnormalities detected

3-3
Male

Killed Day 14

No abnormalities detected

2-0
Female

Killed Day 14

No abnormalities detected

4-0
Female

Killed Day 14

No abnormalities detected

4-1
Female

Killed Day 14

No abnormalities detected

4-2
Female

Killed Day 14

No abnormalities detected

4-3
Female

Killed Day 14

No abnormalities detected

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The acute dermal median lethal dose (LD50) of the test item in the Wistar strain rat was found to be greater than 2000 mg/kg body weight.
Executive summary:

The study was performed to assess the acute dermal toxicity of the test item in the Wistar strain rat.

Methods

Initially, two animals (one male and one female) were given a single, 24 hour, semi-occluded dermal application of the test item to intact skin at a dose level of 2000 mg/kg body weight. Based on the results of the initial test, a further group of eight animals (four males and four females) was equally treated. Clinical signs and body weight development were monitored during the study. All animals were subjected to gross necropsy.

Results

Mortality. There were no deaths.

Clinical Observations. There were no signs of systemic toxicity.

Dermal Irritation. Signs of dermal irritation noted were very slight to well-defined erythema, very slight to slight edema, hemorrhage of dermal capillaries, loss of skin elasticity and flexibility, small superficial scattered scabs and scab cracking.

Body Weight. One female showed no gain in body weight during the first week with expected gain in body weight during the second week and one other female showed expected gain in body weight during the first week but body weight loss during the second week. The remaining animals showed expected gains in body weight over the study period.

Necropsy. No abnormalities were noted at necropsy.


Conclusion

The acute dermal median lethal dose (LD50) of the test item in the Wistar strain rat was found to be greater than 2000 mg/kg body weight. Dermal irritation was low and below threshold for classification. Furthermore, all dermal irritation scores reduced over time, showing the effects being fully reversible.