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Administrative data

Endpoint:
acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 June 2014 to 10 July 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.3 (Acute Toxicity (Dermal))
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test type:
standard acute method
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction Products of Diphosphorus Pentaoxide with Alcohols, C14-18 even, salted with Amines, C12-14, Tert-alkyl
EC Number:
943-540-0
Molecular formula:
Too complex
IUPAC Name:
Reaction Products of Diphosphorus Pentaoxide with Alcohols, C14-18 even, salted with Amines, C12-14, Tert-alkyl
Details on test material:
Description : pale amber liquid
Purity : 100%
Expiry date : 09 May 2016
Storage conditions: room temperature in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Five male and five female Wistar (RccHan™:WIST) strain rats were supplied by Harlan Laboratories UK Ltd., Oxon, UK. On receipt the animals were randomly allocated to cages. The females were nulliparous and non-pregnant. After an acclimatisation period of at least five 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 200g, and were eight to twelve weeks of age.
The animals were housed in suspended solid floor polypropylene cages furnished with woodflakes. The animals were housed individually during the 24 Hour exposure period and in groups of up to four, by sex, for the remainder of the study. Free access to mains drinking water and food (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study. The diet, drinking water and bedding were routinely analysed 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. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve 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:
unchanged (no vehicle)
Details on dermal exposure:
The appropriate amount of test item was applied as evenly as possible to an area of shorn skin (approximately 10% of the total body surface area) using a graduated syringe.
The specific gravity was determined and used to calculate the appropriate dose volume for the required dose level. The specific gravity was 0.917 and the dose volume was 2.19 mL/kg.
Duration of exposure:
24 hours
Doses:
2000 mg/kg body weight
No. of animals per sex per dose:
5 Male
5 Female
Control animals:
not required
Details on study design:
On the day before treatment the back and flanks of each animal were clipped free of hair.
Using available information on the toxicity of the test item, 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 was applied as evenly as possible to an area of shorn skin (approximately 10% of the total body surface area) using a graduated syringe. 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 for the 24 Hour exposure period and for the remainder of the test. 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 dimethyl sulphoxide followed by 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 bodyweight to give a total of five males and five females. After the 24 Hour contact period the bandages were carefully removed and the treated skin and surrounding hair wiped with cotton wool moistened with dimethyl sulphoxide followed by 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 ½, 1, 2 and 4 hours after dosing and subsequently once daily for fourteen days. Due to a technician error, the Day 9 clinical and dermal reaction observations on the initial two treated animals were not performed. This deviation was considered not to affect the purpose or integrity of the study.
After removal of the dressings and subsequently once daily for fourteen days, the test sites were examined for evidence of primary irritation and scored according to the following scale from Draize J H (1977) "Dermal and Eye Toxicity Tests" In: Principles and Procedures for Evaluating the Toxicity of Household Substances, National Academy of Sciences, Washington DC p.31:

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

Oedema Formation

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

Any other skin reactions, if present were also recorded.
Individual bodyweights 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:
No statistical analysis was performed.

Results and discussion

Effect levels
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Remarks on result:
other: 95% confidence limits not reported.
Mortality:
There were no deaths.


Clinical signs:
There were no signs of systemic toxicity.
Body weight:
Animals showed expected gains in bodyweight over the study period.
Gross pathology:
No abnormalities were noted at necropsy.
Other findings:
Dermal Reactions
Very slight erythema with or without very slight edema was noted at the test sites of three females. Other dermal reactions noted at the test sites of females were haemorrhage of the dermal capillaries, crust formation, glossy skin, small superficial scattered scabs. There were no signs of dermal irritation noted at the test sites of males.

Any other information on results incl. tables

Evaluation of Data

Data evaluations included the relationship, if any, between the exposure of the animal to the test item and the incidence and severity of all abnormalities including behavioural and clinical observations, gross lesions, bodyweight changes, mortality and any other toxicological effects.

Using the mortality data obtained, an estimate of the acute dermal median lethal dose (LD50) of the test item was made.

Individual Clinical Observations and Mortality Data

Dose Level

mg/kg

Animal Number and Sex

Effects Noted After Initiation of Exposure (Hours)

Effects Noted After Initiation of Exposure (Days)

½

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

Ä

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 Bodyweights and Weekly Bodyweight Changes

Dose Level mg/kg

Animal Number and Sex

Bodyweight (g) at Day

Bodyweight Change (g) During Week

0

7

14

1

2

2000

1-0 Male

285

303

329

18

26

3-0 Male

266

279

299

13

20

3-1 Male

296

313

335

17

22

3-2 Male

282

299

320

17

21

3-3 Male

256

284

289

28

5

2-0 Female

216

219

222

3

3

4-0 Female

219

222

230

3

8

4-1 Female

220

221

223

1

2

4-2 Female

215

220

226

5

6

4-3 Female

217

221

232

4

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:
other: The acute dermal median lethal dose (LD50) was found to be greater than 2000 mg/kg bodyweight.
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 bodyweight.
Executive summary:

Introduction.

The study was performed to assess the acute dermal toxicity of the test item in the Wistar strain rat. The method was designed to be compatible with the following:

OECD Guidelines for the Testing of Chemicals No. 402 “Acute Dermal Toxicity” (adopted24 February 1987)

Method B3 Acute Toxicity (Dermal) of CommissionRegulation (EC) No. 440/2008

Method

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

Mortality

There were no deaths.

Clinical Observations

There were no signs of systemic toxicity.

Dermal Irritation

Signs of dermal irritation noted at test sites of females included very slight erythema, very slight edema, hemorrhage of the dermal capilliaries, crust formation, glossy skin and small superficial scabs. There were no signs of dermal irritation in the males.

Bodyweight

All animals showed expected gains in body weight.

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 bodyweight.