Lactic acid, compound with 3-[2-(dimethylamino)ethyl] 1-(2-ethylhexyl) toluene-2,4-dicarbamate (1:1)

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29th October 2003 to 3rd February 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted to GLP. This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 425 (Acute Oral Toxicity: Up-and-Down Procedure)

Deviations:

no

GLP compliance:

yes

Test type:

up-and-down procedure

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Lactic acid, compound with 3-[2-(dimethylamino)ethyl] 1-(2-ethylhexyl) toluene-2,4-dicarbamate (1:1)

EC Number:

269-358-5

EC Name:

Lactic acid, compound with 3-[2-(dimethylamino)ethyl] 1-(2-ethylhexyl) toluene-2,4-dicarbamate (1:1)

Cas Number:

68227-46-3

Molecular formula:

C21H35N3O4.C3H6O3

IUPAC Name:

2-hydroxypropanoic acid 2-(dimethylamino)ethyl N-[5-({[(2-ethylhexyl)oxy]carbonyl}amino)-2-methylphenyl]carbamate

Details on test material:

- Name of test material (as cited in study report): WRS-2390TX
- Physical state: Vicous liquid
- Analytical purity: 71.2% in water
- Expiration date of the lot/batch: 15th November 2004
- Storage condition of test material: The test item was stored at ambient laboratory temperature in the dark for the duration of the study.
- Sponsor's information on this batch detailed in Notebook No. 02-181-126.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: They were supplied by Harlan UK Limited, Shaw’s Farm, Blackthorn, Bicester, OX25 1TP, England.
- Age at study initiation: 7-8 weeks old
- Weight at study initiation: 147-217 g on arrival
- Fasting period before study: Food deprivation overnight before dosing. Food was returned 3-4 h after dosing.
- Housing: The animals were housed singly in suspended cages (dimensions 42 x 27 x 20 cm) with stainless steel grid tops and solid bottoms. Wood shavings were used as bedding. The bedding is considered not to contain any additional substances in sufficient concentration to have had any influence on the outcome of the study. Wooden chew sticks were placed in each cage.
Each cage was supplied with a water bottle and metal food hopper.
Cages, cage racks and water bottles were changed weekly during the study.
The floor was cleaned daily and walls and ceiling were cleaned weekly with disinfectant solution (0.5% Tego 2000, Th. Goldschmidt and Company
Limited, Middlesex, England).
- Diet (e.g. ad libitum): Rat and Mouse No. 1 Maintenance Diet, supplied by Special Diets Services Limited, 1 Stepfield, Witham, Essex, CM8 3AD was available ad libitum except for a period of food deprivation overnight before dosing.
- Water (e.g. ad libitum): Water from the domestic mains supply was available ad libitum throughout the study. The water used by Inveresk is analysed by the local water authority for dissolved items, heavy metals, pesticide residues, pH, nitrates and nitrites.
- Acclimation period: The animals were allowed to acclimatise for at least 5 days before dosing.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): During the study, mean environmental maximum and minimum temperatures were 20°C and 19°C respectively.
- Humidity (%): mean relative humidity was 47%.
- Air changes (per hr): minimum of 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): A 12 h light/dark cycle was in operation (light hours 0700-1900 h)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

VEHICLE
- Concentration in vehicle: The test item was administered, using water as the vehicle, at a constant dose volume of 10 mL/kg.
- Justification for choice of vehicle: WRS-2390TX was too viscous to be administered as supplied. Water was
determined at Inveresk to be a suitable vehicle.


MAXIMUM DOSE VOLUME APPLIED:
Limit dose (mg/kg): 2000

DOSAGE PREPARATION (if unusual):
Formulations were prepared on the day of administration, with the exception of that for Animal 14, which was prepared the day before.
Taking into account purity (71.2%), appropriate quantities of WRS-2390TX were weighed and the requisite quantity of water added. Formulations were mixed until visibly homogeneous by sonication.

- Rationale for the selection of the starting dose: The starting dose level was 175 mg/kg based on the calculated LD50 value of
489 mg/kg from a previously conducted cytotoxicity study.

Doses:

175, 550 or 2000 mg/kg

No. of animals per sex per dose:

175 mg/kg = 1
550 mg/kg = 5
2000 mg/kg = 9

Control animals:

no

Details on study design:

- Duration of observation period following administration:
Animals were observed daily for signs of reaction to treatment for up to 14 days after dosing.
- Frequency of observations and weighing: The body weight of each individual animal was recorded on the day of dosing (Day 1), on Days 8 and 15 and at death. The weights at death are not reported but retained in the study data. All animals were examined for reaction to treatment. The onset, intensity and duration of any signs were recorded. Clinical observations were conducted frequently on the day of dosing (Day 1) and once daily thereafter, until kill on Day 15.
- Necropsy of survivors performed: Yes
- Other examinations performed:
All animals, either at death or on Day 15 (at the end of the observation period) were sacrificed by exposure to carbon dioxide and exsanguinated.
The necropsy consisted of an examination of the thoracic and abdominal organs and tissues in situ. All gross lesions were recorded in descriptive terms including location(s), size (in mm), shape, colour, consistency and number. Carcasses were discarded after this procedure.

Statistics:

No formal statistical analysis was conducted. The estimated LD50 was calculated using the AOT425 Statpgm (Version 1.0).

Results and discussion

Preliminary study:

n/a

Effect levels

Sex:

female

Dose descriptor:

LD50

Effect level:

2 000 mg/kg bw

Remarks on result:

other: Estimated

Mortality:

Five out of 9 animals treated with WRS-2390TX at 2000 mg/kg died on Days 2 or 3.

Clinical signs:

At 175 mg/kg, no adverse clinical signs were noted.
At 550 mg/kg, clinical signs were noted in 3 out of 5 animals from approximately 2¼ h after dosing up to Day 2. These included piloerection,
staggering and/or noisy breathing.
At 2000 mg/kg, clinical signs were noted from approximately 30 min after dosing up to the end of the observation period (Day 15). The majority of
clinical signs were noted from approximately 2¾ h after dosing up to Day 2 when 4 of the 5 premature deaths occurred. Predominant clinical signs
included piloerection, cold to touch, laboured breathing, staggering, subdued behaviour, tremors and hunched appearance.

Body weight:

Body weight gain was considered to be satisfactory for animals receiving WRS-2390TX at 175 and 550 mg/kg. The 4 surviving animals treated with
2000 mg/kg had either lost or gained very little weight by Day 8. By Day 15, 2 of these animals (Animals 11 and 14) had lost up to 25% of their Day 1 body weights. This correlated with clinical signs noted up to Day 15 for these 2 animals. The body weight gain for the other 2 animals (Animals 8 and 17) was considered to be satisfactory by Day 15.

Gross pathology:

At 175 and 550 mg/kg, no abnormal findings were noted at necropsy.
At 2000 mg/kg, one animal (Animal 12), which was humanely sacrificed on Day 2, was noted to have its stomach distended by food, liquid and a small amount of gas. One animal (Animal 11), surviving to the end of the observation period, had red staining on the skin around the muzzle.

Other findings:

nda

Any other information on results incl. tables

Mortality was noted as follows:

Test Sequence	Female	Dose Level (mg/kg)	Day of Death	Necropsy
1	6	175	15	Terminal sacrifice
2	7	550	15	Terminal sacrifice
3	8	2000	15	Terminal sacrifice
4	9	2000	3	Found dead
5	10	550	15	Terminal sacrifice
6	11	2000	15	Terminal sacrifice
7	12	2000	2	Humanely sacrificed
8	13	550	15	Terminal sacrifice
9	14	2000	15	Terminal sacrifice
10	15	2000	2	Humanely sacrificed
11	16	550	15	Terminal sacrifice
12	17	2000	15	Terminal sacrifice
13	18	2000	2	Found dead
14	19	550	15	Terminal sacrifice
15	20	2000	2	Found dead

Applicant's summary and conclusion

Interpretation of results:

harmful

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In an Acute Oral Toxicity (Up-and-Down Procedure) Test in Rats (Inveresk report number: 23655) WRS-2390TX was found to have an estimated LD50 of 2000 mg/kg. The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability & repeatability).

Executive summary:

In an Acute Oral Toxicity (Up-and-Down Procedure) Test in Rats (Inveresk report number: 23655) WRS-2390TX was found to have an estimated LD50 of 2000 mg/kg. The estimated LD50 was calculated using the AOT425 Statpgm (Version 1.0).

Five out of 9 animals treated with WRS-2390TX at 2000 mg/kg died on Days 2 or 3. Predominant clinical signs included piloerection, cold to touch, laboured breathing, staggering, subdued behaviour, tremors and hunched appearance.

Clinical signs were noted in 3 out of 5 animals dosed with 550 mg/kg WRS-2390TX from approximately 2¼ h after dosing up to Day 2 and included piloerection, staggering and/or noisy breathing. No clinical signs were noted at 175 mg/kg.

At 2000 mg/kg, 2 of the 4 surviving animals had lost up to 25% of their Day 1 body weights by Day 15. No effects on body weight were observed for the other 2 animals receiving 2000 mg/kg or for animals receiving 175 or 550 mg/kg.

At necropsy, in animals receiving 2000 mg/kg, one animal, which was humanely sacrificed on Day 2, was noted to have its stomach distended by food, liquid and a small amount of gas and one animal surviving to the end of the observation period, had red staining on the skin around the muzzle. No other necropsy findings were observed in any other animals.

The method was determined according to OECD 425 Guideline and is in compliance with the OECD principles of Good Laboratory Practice.