Registration Dossier

Administrative data

Endpoint:
acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Between 12 September 2012 and 03 October 2012
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:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
other: in order to minimise the number of animals required, the study design was based on the OECD 423 Acute Oral Toxicity Study in the Rat – Acute Toxic Class Method, using three animals for each required step.
GLP compliance:
yes (incl. certificate)
Test type:
standard acute method
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Sponsor's identification: 1,1,3,3-tetramethylbutyl hydroperoxide (CAS 5809-08-5)
Description : clear colourless liquid
Batch number : 1010519063
Purity : 91.1%
Date received : 15 November 2011
Expiry date : 01 November 2012
Storage conditions: approximately 4°C in the dark
The integrity of supplied data relating to the identity, purity and stability of the test item is the responsibility of the Sponsor.
A Certificate of Analysis supplied by the Sponsor is given in Appendix 1 (attachment 1)

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Three male and three 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 200 g, and were eight to twelve 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 wood flakes. The animals were housed individually during the 24 hour exposure period and in groups of five, 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 calculated volume of test item, as received, was applied as evenly as possible to an area of shorn skin (approximately 10% of the total body surface area) using a graduated syringe.

Duration of exposure:
24 hours
Doses:
2000 mg /kg body weight
No. of animals per sex per dose:
3 female
3 male
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, a single group of animals was treated as follows:
Dose Level(mg/kg) Specific Gravity Dose Volume(ml/kg) Number of Rats (Female)
2000 0.889 2.25 3
The calculated volume of test item, as received, 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. 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 arachis oil BP to remove any residual test item.
As no mortalities were noted a further group of three males was similarly treated with the test item at a dose level of 2000 mg/kg bodyweight to give a total of three males and three 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 arachis oil BP to remove any residual test item.
The animals were housed in groups of three by sex 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.
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.

Results and discussion

Effect levels
Key result
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:
Individual mortality data are given in Table 1.
There were no deaths.
Clinical signs:
Individual clinical observations data are given in Table 1.
Red/brown staining around the snout was noted thirty minutes after dosing in one male. No other signs of systemic toxicity were noted.
Body weight:
Individual bodyweights and weekly bodyweight changes are given in Table 3.
All animals showed expected gains in bodyweight over the study period.
Gross pathology:
Individual necropsy findings are given in Table 4.
No abnormalities were noted at necropsy.
Other findings:
Dermal Reactions
Individual dermal reactions are given in Table 2.
Very slight erythema was noted at the test sites of all females. Signs of dermal irritation noted at the test sites of both groups were haemorrhage of dermal capillaries, small superficial scattered scabs and crust formation. Other signs of dermal irritation noted at the test sites of females were blanching of the skin, hardened light brown coloured scab, glossy skin and scab lifting to reveal glossy skin.

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.

Table 1              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

Female

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1-1

Female

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1-2

Female

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

2-0

Male

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

2-1

Male

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

2-2

Male

Ss

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0= No signs of systemic toxicity

Ss = Red/brown staining around the snout

Table 2              Individual Dermal Reactions

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

Female

Erythema

1

1

1

1

1

1

1

0

0

0

0

0

0

0

Oedema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Other

Bl

0

0

Hd

Cf

Cf

Cf

0

0

0

0

0

0

0

1-1

Female

Erythema

1

1

1

1

1

1

1

0

0

0

0

0

0

0

Oedema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Other

Bl

0

0

Hd

Cf

CfSs

CfSs

Sp

Sp

Sp

Sp

Sp

Sp

SpSg

1-2

Female

Erythema

1

1

1

1

1

1

1

0

0

0

0

0

0

0

Oedema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Other

Bl

0

0

Hd

HdCf

CfSs

CfSs

Ss

G

G

0

0

0

0

2-0

Male

Erythema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Oedema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Other

0

0

0

0

Cf

CfSs

CfSs

CfSs

CfSs

CfSs

Ss

Ss

Ss

Ss

2-1

Male

Erythema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Oedema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Other

0

0

0

0

Cf

CfSs

CfSs

CfSs

Cf

Cf

Cf

Cf

Cf

Cf

2-2

Male

Erythema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Oedema

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Other

0

0

0

0

CfHd

CfSs

CfSs

CfSs

CfSs

CfSs

CfSs

CfSs

CfSs

Ss

0= No reactions           Bl = Blanching of the skin         Hd = Haemorrhage of dermal capillaries                       Cf = Crust formation         G = Glossy skin

Ss = Small superficial scattered scabs                         Sp = Hardened light brown coloured scab        Sg = Scab lifting to reveal glossy skin

Table 3              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 Female

209

217

222

8

5

1-1 Female

201

219

230

18

11

1-2 Female

223

225

232

2

7

2-0 Male

338

358

380

20

22

2-1 Male

336

340

360

4

20

2-2 Male

330

353

379

23

26

Table 4              Individual Necropsy Findings

Dose Level

mg/kg

Animal Number
and Sex

Time of Death

Macroscopic Observations

2000

1-0 Female

Killed Day 14

No abnormalities detected

1-1 Female

Killed Day 14

No abnormalities detected

1-2 Female

Killed Day 14

No abnormalities detected

2-0 Male

Killed Day 14

No abnormalities detected

2-1 Male

Killed Day 14

No abnormalities detected

2-2 Male

Killed Day 14

No abnormalities detected

Applicant's summary and conclusion

Interpretation of results:
other: 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.
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. At the request of the Sponsor, for ethical reasons and in order to minimise the number of animals required, the study design was based on the OECD 423 Acute Oral Toxicity Study in the Rat – Acute Toxic Class Method, using three animals for each required step.

Method. A group of three females was 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 three males 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. Red/brown staining around the snout was noted thirty minutes after dosing in one male. No other signs of systemic toxicity were noted.

Dermal Irritation. Signs of dermal irritation noted were very slight erythema, blanching of the skin, haemorrhage of dermal capillaries, crust formation, scabbing, glossy skin and scab lifting to reveal glossy skin.

Bodyweight. All animals showed expected gains in bodyweight 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 bodyweight.