Formaldehyde, reaction products with phenol heptyl derivs. and 1,3,4-thiadiazolidine-2,5-dithione

Acute Toxicity: dermal

Administrative data

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 15 February 2012 and 07 March 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Deviations:

no

Test guideline 2

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

Test material information

Constituent 1

Reference substance name:

Reaction product of 1,3,4-thiadiazolidine-2,5-dithione, formaldehyde and phenol, heptyl derivs.

EC Number:

939-460-0

Cas Number:

1471311-26-8

Molecular formula:

Too complex

IUPAC Name:

Reaction product of 1,3,4-thiadiazolidine-2,5-dithione, formaldehyde and phenol, heptyl derivs.

Details on test material:

Sponsor's identification: OS48604M
Description : amber coloured liquid
Batch number : OS48604M
Purity : not supplied
Date received : 13 January 2012
Expiry date : 31 December 2013
Storage conditions: room temperature 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 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.

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:

Individual mortality data are given in Table 1.

There were no deaths.

Clinical signs:

other: Individual clinical observations are given in Table 1. There were no signs of systemic toxicity.

Gross pathology:

Individual necropsy findings are given in Table 5.
No abnormalities were noted at necropsy.

Other findings:

Dermal Reactions
Individual dermal reactions are given in Table 2 and Table 3 - attachment 2
Yellow/brown coloured staining, not preventing evaluation of skin responses, was noted at the test sites of all animals.
Very slight erythema was noted at the test sites of all animals. Other signs of skin irritation noted at the test sites of females were haemorrhage of the dermal capillaries, light brown discolouration of the epidermis, slight desquamation, glossy skin, small superficial scattered scabs, hardened light brown or dark brown/black coloured scab, scab lifting to reveal glossy skin or scab lifting at edges to reveal dried blood. On occasions adverse reactions prevented accurate evaluation of erythema and oedema at the test site of one female. Haemorrhage of the dermal capillaries was also noted at the test site of one male.

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 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	Ä	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

Ä =      Due to a technician error observation not performed

Table 4              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	255	270	303	15	33
	3-0 Male	276	286	301	10	15
	3-1 Male	284	310	322	26	12
	3-2 Male	266	280	298	14	18
	3-3 Male	296	313	326	17	13
	2-0 Female	200	197	209	-3	12
	4-0 Female	205	207	220	2	13
	4-1 Female	208	210	220	2	10
	4-2 Female	219	224	232	5	8
	4-3 Female	234	237	247	3	10

Table 5              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. Very slight erythema was noted at the test sites of all animals. Other signs of skin irritation noted at the test sites of females were haemorrhage of the dermal capillaries, light brown discolouration of the epidermis, slight desquamation, glossy skin, scabbing and scab lifting to reveal glossy skin or dried blood. Haemorrhage of the dermal capillaries was also noted at the test site of one male.

Bodyweight. Animals showed expected gains in bodyweight over the study period, except for one female which showed slight bodyweight loss during the first week but expected gain in bodyweight during the second week.

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.