Naphthenic acids, reaction products with diethylenetriamine

Acute Toxicity: dermal

Administrative data

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-06-08 to 2012-11-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1200 (Acute Dermal Toxicity)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)

Test type:

standard acute method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Naphthenic acids, reaction products with diethylenetriamine

EC Number:

268-610-1

EC Name:

Naphthenic acids, reaction products with diethylenetriamine

Cas Number:

68131-13-5

Molecular formula:

not applicable

IUPAC Name:

Naphthenic acids, reaction products with diethylenetriamine

Details on test material:

Name: Naphthenic acids, reaction products with diethylenetriamine
CAS No.: 68131-13-5
Batch No.: ESD0011831
Certificate of Analysis: 26.05.2011 (see Appendix)
Chemical Name: Naphthenic acids, reaction products with diethylenetriamine
Physical State at RT: liquid (highly viscous)
Density: 0.978 g/cm3 (20°C, DIN EN ISO 12185)
Colour: brown
Expiry Date: 04/2013
Purity: 100% (UVCB substance)
Storage Conditions: at room temperature
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Full barrier in an air-conditioned room
- Temperature: 22 +/- 3 °C
- Relative humidity: 55 +/- 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (lot no. XX)
- Free access to tap water, sulphur acidified to a pH value of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- The animals were kept individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding (lot no. XX)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least five days) under laboratory conditions

Administration / exposure

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Details on dermal exposure:

Preparation of the Animals:
The animals were marked for individual identification by tail painting.
Approximately 24 hours before the test, the fur was removed from the dorsal area of the trunk using an electric clipper. Care was taken to avoid abrading the skin, and only animals with healthy intact skin were used.
No less than 10% of the body surface was cleared for the application.
Prior to the application a detailed clinical observation was made of all animals.
Application:
The test item was applied at a single dose, uniformly over an area which was approximately 10% of the total body surface.
The test item was held in contact with the skin by a dressing throughout a 24-hour period. The dressing consisted of a gauze-dressing and non-irritating tape and was fixed with an additional dressing in a suitable manner.

Duration of exposure:

The test item was held in contact with the skin throughout a 24-hour period. At the end of the exposure period the residual test item was removed.

Doses:

The test item was applied at a single dose of 2000 mg/kg body weight to each animal.

No. of animals per sex per dose:

5 male and 5 female

Control animals:

not required

Details on study design:

Observation period:
All animals were observed for 14 days after dosing

Weight Assessment:
The animals were weighed on day 1 (prior to the application) and on days 8 and 15.

Clinical Examination:
careful clinical examination was made several times on the day of dosing (at least once during the first 30 minutes and with special attention given
during the first 4 hours post-dose). As soon as symptoms were noticed they were recorded. Thereafter, the animals were observed for clinical signs once daily until the end of the observation period. All abnormalities were recorded.
Cageside observations included changes in the skin and fur, eyes and mucous membranes. Also respiratory, circulatory, autonomic and central
nervous systems and somatomotor activity and behaviour pattern were examined. Attention was directed to observations of tremors, convulsions,
salivation, diarrhoea, lethargy, sleep and coma.

Pathology:
At the end of the observation period the surviving animals were sacrificed with an overdosage of pentobarbital injected intraperitoneally (Narcoren®, Merial) at the dosage of approximately 8 mL/kg bw. All animals were subjected to gross necropsy. All gross pathological changes were recorded and in case of findings the tissues were preserved for a possible histopathological evaluation. The preserved tissues of which no histopathological
evaluation was made will be discarded 3 months after the release of the final report unless otherwise agreed upon with the sponsor.

Evaluation of Results:
Individual reactions of each animal were recorded at each time of observation.
Toxic response data were recorded by sex and dose level.
Nature, severity and duration of clinical observations were described.
The body weight changes were summarised in a tabular form.
Necropsy findings were described.

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the
results is not regarded as necessary.

Results and discussion

Effect levels

Sex:

male/female

Dose descriptor:

LD0

Effect level:

2 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortality was observed

Clinical signs:

other: No treatment-related effects were observed.

Gross pathology:

No treatment-related effects were observed.

Other findings:

Erythema grade 1 and 2 were observed in 5 of 5 male and 5 of 5 female animals. Oedema grade 1 was observed in 3 of 5 male and 4 of 5 female
animals. Oedema grade 2 was observed in 1 of 5 female animals. Eschar was observed in 5 of 5 male and 5 of 5 female animals. Wound was
observed in 2 of 5 male and 3 of 5 female animals. Necrosis was observed in 1 of 5 female animals.
All signs of irritation were not fully reversible within the observation period.

Any other information on results incl. tables

Table Clinical Signs of Systemic Toxicity – Individual Data - Males:

Animal No. / Sex / Dose	Time of Observation (Post-Dose)	Observations (for Signs of Dermal Irritation, see Table 5)
21/ male / 2000 mg/kg bw	during the whole observation period	no signs of toxicity
22/ male / 2000 mg/kg bw	during the whole observation period	no signs of toxicity
23/ male / 2000 mg/kg bw	during the whole observation period	no signs of toxicity
24/ male / 2000 mg/kg bw	during the whole observation period	no signs of toxicity
25/ male / 2000 mg/kg bw	during the whole observation period	no signs of toxicity

min = minute(s), h = hour(s), bw = body weight

Table Clinical Signs of Systemic Toxicity – Individual Data – Females:

Animal No. / Sex / Dose	Time of Observation (Post-Dose)	Observations (for Signs of Dermal Irritation, see Table 6)
26/ female / 2000 mg/kg bw	during the whole observation period	no signs of toxicity
27/ female / 2000 mg/kg bw	during the whole observation period	no signs of toxicity
28/ female / 2000 mg/kg bw	during the whole observation period	no signs of toxicity
29/ female / 2000 mg/kg bw	during the whole observation period	no signs of toxicity
30/ female / 2000 mg/kg bw	during the whole observation period	no signs of toxicity

min = minute(s), h = hour(s), bw = body weight

Table Skin Irritation – Individual Data – Males:

Day after Start of Application	Animal No. 21		Animal No. 22		Animal No. 23		Animal No. 24		Animal No. 25
	E/O	Comments	E/O	Comments	E/O	Comments	E/O	Comments	E/O	Comments
day 2	2/0	nsf	1/0	nsf	2/1	nsf	2/1	nsf	2/1	nsf
day 3	2/0	nsf	2/0	nsf	2/1	nsf	2/1	nsf	2/1	nsf
day 4	2/0	nsf	2/0	nsf	2/1	nsf	2/1	nsf	2/1	nsf
day 5	2/0	nsf	2/0	nsf	2/1	nsf	2/1	nsf	2/1	Es
day 6	2/0	Es	2/0	Es	2/0	Es	2/0	Es, W(10)	2/0	Es, W(15)
day 7	2/0	Es	2/0	Es	2/0	Es	2/0	Es, W(10)	2/0	Es, W(10)
day 8	1/0	Es	2/0	Es	2/0	Es	1/0	Es	1/0	Es
day 9	1/0	Es	2/0	Es	2/0	Es	1/0	Es	1/0	Es
day 10	1/0	Es	1/0	Es	2/0	Es	1/0	Es	1/0	Es
day 11	1/0	Es	1/0	Es	2/0	Es	1/0	Es	1/0	Es
day 12	1/0	Es	1/0	Es	2/0	Es	1/0	Es	1/0	Es
day 13	1/0	Es	0/0	Es	1/0	Es	1/0	Es	1/0	Es
day 14	1/0	Es	0/0	Es	1/0	Es	1/0	Es	1/0	Es
day 15	1/0	Es	0/0	Es	1/0	Es	1/0	Es	1/0	Es
Comments: E = erythema; O = oedema; 1, 2, 3, 4 = scoring system laid down in OECD Guideline 404 (Table 2) Es = eschar; W (Ømm) = wound; nsf = no specific findings

Table Skin Irritation – Individual Data – Females:

Day after Start of Application	Animal No. 26		Animal No. 27		Animal No. 28		Animal No. 29		Animal No. 30
	E/O	Comments	E/O	Comments	E/O	Comments	E/O	Comments	E/O	Comments
day 2	2/0	nsf	2/1	nsf	1/0	nsf	2/0	nsf	2/1	nsf
day 3	2/1	nsf	2/2	nsf	1/0	nsf	2/1	*	2/1	nsf
day 4	2/1	nsf	2/2	nsf	1/0	nsf	2/1	*	2/1	nsf
day 5	2/1	nsf	2/0	Es, W (2 cm)	1/0	nsf	2/1	*	2/1	nsf
day 6	2/0	N (15)	2/1	Es, W (2 cm)	2/0	Es, W (15 mm)	2/1	Es, *	2/0	W (2.5 cm)
day 7	2/0	N (15)	2/1	Es, W (2 cm)	2/0	Es, W (15 mm)	2/1	Es, *	2/0	W (2.5 cm)
day 8	2/0	N (15)	2/0	Es	2/0	Es	2/1	Es, *	2/0	W (2.5 cm)
day 9	2/0	Es, N (15)	2/0	Es	2/0	Es	2/0	Es, *	2/0	W (2.5 cm)
day 10	2/0	Es, N (15)	2/0	Es	2/0	Es	2/0	Es, *	2/0	Es, W (0.5 cm)
day 11	2/0	Es, N (15)	2/0	Es	2/0	Es	2/0	Es	2/0	Es, W (0.5 cm)
day 12	2/0	Es, N (15)	2/0	Es, W (2x4cm)	2/0	Es	2/0	Es	2/0	Es
day 13	1/0	Es	2/0	Es, W (2x4cm)	1/0	Es	1/0	Es	1/0	Es
day 14	1/0	Es	1/0	Es, W (1x2cm)	1/0	Es	1/0	Es	1/0	Es
day 15	1/0	Es	1/0	Es	1/0	Es	1/0	Es	1/0	Es
Comments: E = erythema; O = oedema; 1, 2, 3, 4 = scoring system laid down in OECD Guideline 404 (Table 2) Es = eschar; N (Ømm) = necrosis; W (Ø) = wound; nsf = no specific findings; * = animal very nervous

Table Absolute Body Weights in g and Body Weight Gain in %:

Dose: 2000 mg/kg body weight
Animal No. / Sex	g Day 1	g Day 8	g Day 15	% Day 1-15
21 / male	238	243	276	16
22 / male	245	262	291	19
23 / male	232	220	249	7
24 / male	231	230	261	13
25 / male	241	238	261	8
26 / female	223	220	220	-1
27 / female	211	198	209	-1
28 / female	210	205	215	2
29 / female	221	214	220	0
30 / female	212	205	216	2

Table Macroscopic Findings - Individual Data – Males and Females:

Dose: 2000 mg/kg bw
Animal No. / Sex	Organ	Macroscopic Findings
21 / male	-	nsf
22 / male	-	nsf
23 / male	-	nsf
24 / male	-	nsf
25 / male	liver	liver partially located in thorax
26 / female	-	nsf
27 / female	-	nsf
28 / female	-	nsf
29 / female	-	nsf
30 / female	-	nsf

nsf = no specific findings

Table LD50:

Dose (Unit)	Number of Animals Investigated	Number of Intercurrent Deaths	LD50
2000 mg/kg bw	5 males	0	> 2000 mg/kg bw
2000mg/kg bw	5 females	0	> 2000 mg/kg bw

bw = body weight

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Remarks:

Migrated information

Conclusions:

Under the conditions of the present study, single dermal application of NA-DETA to rats at a dose of 2000 mg/kg body weight was associated with neither mortality nor signs of toxicity but signs of irritation.
The dermal LD50 was determined to be > 2000 mg/kg body weight.

Executive summary:

 The acute dermal toxicity of NA-DETA was investigated according to the Guideline OECD 402.

NA-DETA was applied dermally to rats at dose level of 2000 mg/kg body weight. No mortality, no signs of toxicity was observed.

The dermal LD50 was determined to be > 2000 mg/kg body weight.