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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
other: subacute
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October-November 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted study according to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to
Guideline:
other: MHW-MITI (1986)
Deviations:
not specified
Principles of method if other than guideline:
A 28 day oral gavage study was performed with the test item in maize oil on 5 male and 5 female CD rats per dose at dosages of 0, 10, 50 and 200 mg/kg bw/day according to OECD 407 in 1993. Satellite groups of 5 rats/sex/dose were simultaneously treated with 0 (vehicle alone) and 200 mg/kg bw/day to assess the reversibility of effects.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): BCI-MX
Batch no: 910312 (see CoA attached)
Purity: 88±3% (m/m)
Other: mono-citracon-mono-itaconimide isomer: 6.5±2% (m/m)
Unknown: 5.5±2% (m/m)
appearance: creme-white powder

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
maize oil
Details on oral exposure:
Method of administration: gavage (5ml/kg bw)
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
Test duration: 28 days
Frequency of treatment:
Dosing regime: daily
Doses / concentrations
Remarks:
Doses / Concentrations:
10, 50, 200 mg/kg bw
Basis:
other: nominal in maize oil
No. of animals per sex per dose:
5 per sex per dose plus
5 animals/sex of the control and high dose group were treated for 28 days and then kept for a 14-day recovery period.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
3 rats/sex and dose were treated with 200, 500, or 1000 mg/kg bw and day for 7 consecutive days.
A single male treated at 1000 mg/kg bw/d and a single female rat treated at 500 mg/kg bw/d were underactive, ungroomed, piloerect and cold on day 1. There were both found dead on the following day.
The remaining rats treated at 1000 mg/kg were sacrificed at day 3 for humane reasons, since they showed the same signs as the animals described above including body weight loss.
In the 500 mg/kg bw group piloerection, post dose salivation etc. were seen from day 1 until termination. The majority of the animals lost bw during the first 3 days of treatment, but gained weight satisfactorily thereafter.
At 200 mg/kg and day piloerection, post-dose salivation and ungroomed conditions were seen. The animals achieved satisfactory body weight gains.
Accordingly the doses for the main study were chosen with 10, 50 and 200 mg/kg bw, the latter being considered to be at or close to the maximum tolerated soage under these conditions of treatment.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: on the day of treatment and twice weekly afterwards

FOOD CONSUMPTION : Yes
Food consumption ratio: yes

WATER CONSUMPTION: Yes (visually)

OPHTHALMOSCOPIC EXAMINATION: No (not required at the time of study)

HAEMATOLOGY: Yes

Blood CHEMISTRY: Yes

URINALYSIS: Yes

NEUROBEHAVIOURAL EXAMINATION: No (not required at the time of study)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes (kidneys, liver spleen, adrenals, heart, for all animals of the main groups and the reversibility groups; addtionally the stomach was histopathologically investigated since abnormal findings were obsered in this organ)
Statistics:
Inter-group differences in group mean bodyweight change, haematology, blood chemistry and Quantitative urinalysis were evaluated by Student's 't'-test using a pooled error variance. The results of this test are not presented for eosinophil, basophil or monocyte counts where the data are clearly not normally distributed. For organ weights, homogeneity of variance was tested using Bartlett's test. If this was found to be statistically significant, a Behrens-Fisher test was used to perform pairwise comparisons, otherwise Dunnett's test was used. Inter-group differences in the incidence of macro- or micropathological lesions were assessed by the Fisher Exact Probability test. This is in line with current practice but differs from the protocol. Two-tailed analyses were undertaken unless otherwise indicated.
Levels of statistical significance were chosen as p < 0.05 (a), p < 0.01 (b) and p < 0.001 (c) for Student's 't'-test and p < 0.05 (a) and p < 0.01 (b) for Dunnett's or Behrens-Fisher tests and the Fisher Exact Probability test. Inter-group differences that were not statistically significant (p > 0.05) are not annotated.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Decedents: Signs included hunched posture, ungroomed appearance, piloerection, and single observations of loose faeces, pallor of eyes, salivation, hypothermia and prostration.
Survials: Signs of reaction to treatment in surviving animals treated at 200 mg/kg/day were minor and generally transient: salivation from Day 5, ungroomed fur on occasions from Day 6, isolated incidences of loose faeces on Days 10 and 11 and piloerection on Days 24 and 25. One male rat had rales between Days 12 and 14 and also showed piloerection and hunched posture on Day 12.
Signs of reaction to treatment in animals treated at 50 mg/kg/day were similarly minor: salivation fro Day 6 and isolated incidences of piloerection, ungroomed fur, hunched posture, rales and laboured respiration.
Three ats treated at 10 mg/kg/day showed salivation on Days 25 and 30.
Mortality:
mortality observed, treatment-related
Description (incidence):
Four males and one female treated at 200 mg/kg/day were found dead or humanely killed between Days 9 and 26.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The bodyweight gains at 200 mg/kg/day were slightly lower than those of the control animals during the first two weeks of treatment.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The food consumption at 200 mg/kg/day were slightly lower than those of the control animals during the first two weeks of treatment.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
At 200 mg/kg/day a commensurate reduction in food utilization efficiency was recorded.
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Aspartate amino-transferase activities and plasma urea concentrations of male rats treated at 200 mg/kg/day and killed after four week of treatment ere slightly higher than those of control males. These differences were not observed at the end of the two week recovery period.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Urinary protein concentrations of two male rats treated at 200 mg/kg/day an killed after four weeks of treatment were noticeably higher than those of control rats, Reducing substances and glucose were detected in the urine of further two male rats treated at this dosage. These differences were not observed ten days after treatment had ceased.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Organ weights were considered to have been unaffected by treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There was no macropathological finding amongst animals killed after four weeks of treatment. Thickened stomach wall was recorded for one female rat which received 200 mg/kg/day and was killed after two weeks cessation of treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Hyperkeratosis an acanthosis in the keratinised region of the stomach were observed in animals treatet at 200 mg/kg/day. All decedents and most animals receiving this dosage and killed after four weeks of treatment showed these findings. Although the findings were still evident in animals two weeks after treatment had ceased, their frequency was much reduced.
Histopathological findings: neoplastic:
not examined
Details on results:
Mortality: Four male and one female animal from the high dose group were foud dead or humanely killed. Ante mortem signs were
rales, gasping and abdominal distension, hunched posture and piloerection. Histopathology revealed that all of these animals had hyperkeratosis and acanthosis of the keratinised region of the stomach and one male animal had accomanying ulceration. The ill-health or demise of these animals was therefore considered to be, at least in part, caused by an irritant effect of the test material on the stomach wall.
Clinical signs:The surviving animals of the high dose group showed salivation, ungroomed appearance and piloerection. The mid-dose animals showed salivation and isolated incidences of pilo-erection, hunched posture and ungroomed fur. Of the low dose group three rats showed salivation.
Body weight: slightly reduced at 200 mg/kg bw
Laboratory findings: Haematology was unaffected. In the high dose group aspartate asmino-tranferase activity and plasma urea concentration were slightly higher in the animals killed at day 29. These differences were not observed at the end of the 2-week reversibility period.
Effects in organs: No treatment related effects on organ weights or macropathological effects observed. Thickened stomach was recorded for one female rat in the high dose group which was humanely killed.
Histopathology: In the rats from the high dose level, hyperkeratosis and acanthosis in the keratinised region of the stomach were seen. Although the findings were still evident in animals 2 weeks after treatment had ceased, their frequency was much reduced.
Hyperkeratosis and acanthosis of the stomach were associated with the repeated gavage adiministration of the test compound. The presence of these findings in the high dosage group is likely to be the result of an irritant effect of the test material rather than a toxic effect. Inflammatory effects occured more prominent in the stomach of high dosed animals.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on irritating effects in the stomach and its consequences at 200 mg/kg bw/day

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
50 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
yes

Applicant's summary and conclusion

Conclusions:
The NOAEL in this study is 50 mg/kg bw/day based on irritating effects in the stomach and its consequences at 200 mg/kg bw/day. The NOEL was considered to be very close to this dosage.
Executive summary:

A 28 day oral gavage study was performed with the test item in maize oil on 5 male and 5 female CD rats per dose at dosages of 0, 10, 50 and 200 mg/kg bw/day according to OECD 407 in 1993. Satellite groups of 5 rats/sex/dose were simultaneously treated with 0 (vehicle alone) and 200 mg/kg bw/day to assess the reversibility of effects.

The dose of 200 mg/kg bw/day group resulted in death of four males and one female. Clinical signs and histopathology (all of these animals had hyperkeratosis and acanthosis of the keratinised region of the stomach and one had accompanying ulceration) indicated that these deaths were, at least in part, caused by an irritant effect of the test material on the stomach wall. Most survivors also showed hyperkeratosis and acanthosis and a higher incidence of acute inflammation of the glandular and keratinised stomach regions compared with contol animals. After two weeks recovery, the proportion of animals affected was much lower. A slightly reduced body weight gain was seen in the high dose group and clinical signs as salivation after dosing, ungroomed appearance and piloerection.

At 50 mg/kg bw/day the clinical signs were similar but minor and nearly dissapeared at 10 mg/kg bw.

Hyperkeratosis and acanthosis of the stomach were associated with gavage administration of the test compound. The presence of these findings in the high dosage group is likely to be the result of an irritant effect of the test material rather than a toxic effect. The NOAEL in this study is 50 mg/kg bw/day based on irritating effects in the stomach and its consequences at 200 mg/kg bw/day. The NOEL was considered to be very close to this dosage.