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Toxicological information

Acute Toxicity: oral

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Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 1983 to May 1983
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was performed in accordance with OECD test guideline 401 Acute Oral Toxicity and in compliance with Good Laboratory Practice regulations.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1983
Report Date:
1983

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 401 (Acute Oral Toxicity)
GLP compliance:
yes
Test type:
acute toxic class method
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
The test article was a yellow-brown liquid supplied in a glass screw-capped bottle labelled Diethyl-Toluene-diamine. The test article was stored at room temperature in the dark.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Ltd, Manston Road, Margate, Kent, U.K.
- Age at study initiation: The animals were approximately 7 to 11 weeks old at study initiation.
- Weight at initiation of definitive study: males (149 - 177 grams), females (125 - 142 grams)
- Fasting period before study: Yes
- Housing: Solid floor polypropylene cages were used, which contained a bedding of softwood saw dust (Sawdust Marketing Company Ltd., Standon Herts.) that was replaced twice weekly. Animals were caged in groups of 4 by sex for the screening study and in groups of 5 by sex and dose group for the definitive study
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 °C to 21 °C
- Humidity (%): 52 to 70%
- Photoperiod (hrs dark / hrs light): 12 hrs dark/ 12 hrs light


IN-LIFE DATES: From: April 1983 To: May 1983

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
VEHICLE
- Concentration in vehicle: The following concentrations were used to achieve the respective dose levels in the screening study: 5 mg/ml (50 mg/kg), 25 mg/ml, (250 mg/kg), 125 mg/ml (1250 mg/kg), and 500 mg/ml (5000 mg/kg). For the definitive study, a concentrated solution of 100 mg/ml wadministered at different volumes, discussed below, to achieve the following dose levels: 500 mg/kg, 707 mg/kg, 1000 mg/kg, and 1410 mg/kg.
- Amount of vehicle (gavage): The screening study utilized a dose volume of 10 ml/kg. The definitive study utilized the following dose volumes: 5 ml/kg (500 mg/kg), 7.07 ml/kg (707 mg/kg), 10.00 ml/kg (1000 mg/kg), and 14.10 ml/kg (1410 mg/kg).
- Justification for choice of vehicle: Corn oil is a standard vehicle used for toxicological studies, and there was no information to indicate that the stability of the test article would be adversely affected under the proposed conditions of administration.

MAXIMUM DOSE VOLUME APPLIED: The maximum dose volume applied was 2.20 ml in the 1410 mg/kg group from the definitive study.

DOSAGE PREPARATION (if unusual): Fresh formulations of the test article were made for the screening and definitive studies.
Doses:
The dose levels used in the screening study were 50 mg/kg, 250 mg/kg, 1250 mg/kg, and 5000 mg/kg. For the definitive study, the following dose levels were used: 500 mg/kg, 707 mg/kg, 1000 mg/kg, and 1410 mg/kg.
No. of animals per sex per dose:
The screening study employed two fasted rats (1 male, 1 female) per dose level. The definitive study employed ten fasted rats (5 males, 5 females) per dose group.
Control animals:
no
Details on study design:
- Allocation of animals to treatment groups: The animals were allocated to treatment groups by means of a total randomization procedure. Animals were transferred as they came to hand from the delivery crates to holding cages starting at the left side of the top row of the battery and working left to right and top to bottom, until each cage contained one animal. This procedure was repeated until each cage contained 4 animals (screening study) or 5 animals (definitive study). Treatment groups were assigned to the cages using a set of random letter permutations.
- Duration of observation period following administration: For the screening study, animals were observed for 48 hours post dosing. Animals in the definitive study were observed for 14 days post dosing.
- Frequency of observations and weighing: Animals in the screening study were weighted on the day of treatment to allow the calculation of individual treatment volumes. For the definitive study, individual body weights were recorded on the day before treatment (day -1), on the day of treatment, and on day 7 and 14 after treatment and at death.
- Necropsy of survivors performed: No necropsies were performed in the screening study. In the definitive study, all animals were subjected to a gross necropsy. Animals surviving the 14 day observation period were killed by carbon dioxide asphyxiation.
- Other examinations performed: All animals in the definitive study were observed for overt signs of toxicity or behavioral changes at 1/4, 1, 2, and 4 hours post dosing and subsequently once daily for 14 days. All gross or visible toxic or pharmacological effects were recorded.
Statistics:
The acute oral median lethal dose (LD50) for combined male and female groups were calculated using a probit analysis (Finney 1964). Separate LD50 values were calculated for male and female animals. The mortalities did not allow the calculation of 95% fiducial limits or the production of a dose response curve.

Finney, D.J. (1964) Statistical Method for Biological Assay, 2nd Edition, Charles Giffin, London.

Results and discussion

Effect levelsopen allclose all
Sex:
male/female
Dose descriptor:
LD100
Effect level:
>= 1 000 mg/kg bw
Remarks on result:
other: Definitive study
Sex:
female
Dose descriptor:
approximate LD50
Effect level:
ca. 755 mg/kg bw
Remarks on result:
other: Definitive study, LD50 calculated by probit method
Sex:
male/female
Dose descriptor:
approximate LD50
Effect level:
ca. 738 mg/kg bw
Remarks on result:
other: Definitive study, LD50 calculated by probit method
Sex:
male
Dose descriptor:
approximate LD50
Effect level:
ca. 723 mg/kg bw
Remarks on result:
other: Definitive study, LD50 calculated by probit method
Sex:
male/female
Dose descriptor:
other: 30% mortality
Effect level:
>= 707 mg/kg bw
Remarks on result:
other: Definitive study (2/5 males, 1/5 females died)
Sex:
male/female
Dose descriptor:
LD0
Effect level:
> 500 mg/kg bw
Remarks on result:
other: Definitive study (5 animals/sex/group)
Sex:
male/female
Dose descriptor:
LD0
Effect level:
> 250 mg/kg bw
Remarks on result:
other: Screening study (1 animal/sex/group)
Sex:
male/female
Dose descriptor:
LD0
Effect level:
> 50 mg/kg bw
Remarks on result:
other: Screening study (1 animal/sex/group)
Mortality:
In the screening study, the mortalities indicated an LD50 in the range of 250 - 1250 mg/kg. Neither male or female rat in the 50 or 250 mg/kg group died (0% mortality); however, both male and female rats in the 1250 and 5000 mg/kg groups died (100% mortality). In the definitive study, a total of 23 (12 male, 11 female) of the 40 animals died during the study period. All deaths, with one exception, were noted 24 or 48 hours after treatment. One animal treated with 1000 mg/kg was found dead on day 6.
Clinical signs:
Before initiating the definitive study, all animals were examined for signs of ill health or injury. All animals appeared healthy and no animals were discarded.

All animals treated with 500 mg/kg appeared normal throughout the study period.

The major sign of toxicity noted in animals treated with 707 mg/kg during the day of dosing was lethargy. One animal showed signs of piloerection at this time. The day after dosing all animals showed lethargy, spasticity of the hind limbs, hunched posture and fully closed eyes. Piloerection and chromodacryorrhoea were also occasionally noted. Surviving animals appeared normal on day 6 and throughout the study period.

Animals treated with 1000 mg/kg appeared lethargic with piloerection on the day of dosing. The day after dosing all animals showed hunched posture and fully closed eyes. Occasional signs of piloerection, ataxia, increased sensitivity to touch, aggressiveness when touched and chronic convulsions were noted at this time. The one surviving animal showed piloerection, fully closed eyes, ataxia and high stepping gait 48 hours after treatment and appeared lethargic until it was found dead on day 6.

All animals treated with 1410 mg/kg were lethargic with piloerection during the day of dosing. Surviving animals showed hunched posture, fully closed eyes and ataxia 24 hours after dosing. All animals were dead 48 hours after treatment.
Body weight:
Male rats in the 500 mg/kg of the definitive study gained an average of 86 grams of body weight postdosing up to day 14. The surviving male rats (3/5) in the 707 mg/kg group gained an average of 78 grams of body weight postdosing up to day 14.

Female rats in the 500 mg/kg group of the definitive study gained an average of 53 grams of body weight postdosing up to day 14, compared to the average weight gain of 43 grams for the surviving animals (4/5) in the 707 mg/kg group.

Gross pathology:
The major pathological findings in animals dying during the definitive study were associated with the stomach, which appeared distended. The gastrointestinal tract was occasionally filled with gas and the liver of one animal showed pale patches.

No abnormalities were noted in animals necropsied at the end of the study period.

Applicant's summary and conclusion

Conclusions:
The calculated LD50 for all animals (male and female) was 738 mg/kg.
Executive summary:

The acute oral median lethal dose (LD50) was calculated by a probit method. The values obtained were: All animals (738 mg/kg), male animals (723 mg/kg), and female animals (755 mg/kg). The mortalities did not allow the calculation of 95% fiducial limits.