Registration Dossier

Administrative data

Endpoint:
methaemoglobinaemia
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From March 18 to 19, 1981.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Test procedures cannot be subsumed under a testing guideline, nevertheless are well documented and scientifically acceptable.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1981
Report Date:
1981

Materials and methods

GLP compliance:
no
Type of method:
in vivo
Endpoint addressed:
acute toxicity: oral

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
other: Tif: RAIf
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: test laboratory.
- Age at study initiation: young adult rats, age not specified.
- Weight at study initiation: 180 - 250 g.
- Fasting period before study: overnight.
- Housing: two in Macrolon Cages, Type 3 with wire mesh tops and saw dust bedding (granular form).
- Diet : ad libitum, Rat chow N. 890 (NAFAG, Gossau SG).
- Water: ad libitum.
- Acclimation period: ad least 7 days.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 55 ± 10 %
- Photoperiod: 14/10 hrs dark / hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
The test substance was dissolved in tap water in order to achieve a dosage level of 20 ml/kg body weight.
Prior to the administration the formulation was homogeneously dispersed with an Ultra-Turrax and subsequently placed on a magnetic stirrer.

Analytical verification of doses or concentrations:
not specified
Frequency of treatment:
Once.
Post exposure period:
24 hours.
Doses / concentrations
Remarks:
Doses / Concentrations:
5000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
8 animals in control group.
4 animals in the treated group and in the positive control group.
Control animals:
yes, concurrent vehicle

Examinations

Examinations:
CLINICAL SIGNS and MORTALITY
Symptoms and mortality were assessed 1, 2, 4, 6 and 24 hours after the administration.

BLOOD SAMPLING
Blood was collected by puncture of the retroorbital plexus using heparinized micro-haematocrit tubes at the following times:
- prior to the administration (all animals);
- 15, 30, 60, 90 minutes, 2, 4, 6 and 24 hours (each time half group).
The blood samples were immediately cooled and the haematological investigation was performed without delay.
Positive control:
The identical test was performed with a positive control group which received 40 mg/kg sodium nitrite solved in tap water, administered at a rate of 20 ml/kg.

Results and discussion

Details on results:
SYMPTOMS
Sedation, dyspnea, ruffled fur, diarrhea and curved body position were observed.

MORTALITY
No animal died during the test period.

METHAEMOGLOBIN-induction
The individual and mean values of the Methaemoglobin determinations are listed in Table 2 - 4.

Any other information on results incl. tables

Statistical Analysis of Methaemoglobin values

Generally the mean values of the test group are inferior to the ones of the negative control group with the exception of the samples taken after 15 min., 90 min. and 6 hours.

As the overall mean of the negative control group is superior to the one of the test group, no significant increase of Methaemoglobin can be demonstrated.

Table 1A: Symptoms observed in the negative control group

Symptoms Observation time after administration (hours)
1 2 4 6 24
ruffled fur +

Table 1B: Symptoms observed in the test group

Symptoms Observation time after administration (hours)
1 2 4 6 24
sedation + + + + +
dyspnea + + +
ruffled fur + + + + +
diarrhea + +
curved body position + + + +

Table 1C: Symptoms observed in the postive control group

Symptoms Observation time after administration (hours)
1 2 4 6 24
sedation ++ ++ + + +
dyspnea + + + + +
ruffled fur ++ ++ ++ ++ ++
curved body position + + + + +

key: + = slight, ++ = moderate, +++ = severe

Table 2: Methaemoglobin levels in negative control group:

Time animals (n) mean standard deviation
pretest 16 0.65 0.186
15 min 8 0.638 0.25
30 min 7 0.542 0.181
60 min 8 0.738 0.288
90 min 8 0.638 0.192
2 h 8 0.675 0.149
4 h 8 1.038 0.297
6 h 8 0.925 0.324
24 h 8 1.2 0.428

Table 3: Methaemoglobin levels in test group:

Time animals (n) mean standard deviation
pretest 8 0.538 0.262
15 min 4 0.7 0.432
30 min 4 0.475 0.359
60 min 4 0.55 0.412
90 min 4 1.075 0.299
2 h 4 0.5 0.258
4 h 4 0.7 0.837
6 h 4 1.225 0.506
24 h 4 0.75 0.342

Table 4: Methaemoglobin levels in positive control group:

Time animals (n) mean standard deviation
pretest 8 0.7 0.2
15 min 4 17.8 3.2
30 min 4 19.8 12.1
60 min 3 30.6 6.1
90 min 4 23.2 16.9
2 h 4 24.4 13.6
4 h 4 4.1 4.9
6 h 4 0.8 0.4
24 h 4 1 0.6

Applicant's summary and conclusion

Conclusions:
The test substance has been found to be devoid of Methaemoglobin-inducing capacity after single oral administration of 5000 mg/kg in young adult rats.
Executive summary:

Method

The purpose of the study was evaluate the Methaemoglobin-inducing capacity of the test substance in comparison with a negative control.

Results

The test substance has been found to be devoid of Methaemoglobin-inducing capacity after single oral administration of 5000 mg/kg in young adult rats.