Reaction product of ammonium molybdate and C12-C24-diethoxylated alkylamine (1:5-1:3)

Endpoint summary

Description of key information

Repeated dose toxicity (oral route): No Observed Effect Level (NOEL) 200 mg/kg/day (OECD 407, GLP study)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 June 1993 to September 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL Ltd., Basel, Switzerland
- Age at study initiation: Approx. 6 weeks
- Weight at study initiation: all animals within ± 20% of the sex mean
- Housing: Group housing of 5 animals per sex per cage in stainless steel suspended cages with wire mesh floors.
- Diet (e.g. ad libitum): Free access to standard pelleted laboratory animal diet (Kliba 343 from Klingentalmuhle AG, Kaiseraugst, Switzerland).
- Water (e.g. ad libitum): Free access to tap—water
- Acclimation period: At least 5 days before start of treatment under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): relative humidity of 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): Lighting was 12 hours artificial fluorescent light and 12 hours dark per day.

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

The test substance was administered to rats by daily oral gavage for a period of 28 days, followed by a 14-day recovery period.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of formulations prepared during week 4 and after treatment (using identical procedures to those used during the treatment period) were
analysed using HPLC to check stability, homogeneity and accuracy of preparation.

SPECTROPHOTOMETRY CONDITIONS:
Bandpass: 2 nm
Detection wavelength: 319 nm
Temperature: room temperature
Cuvettes: quarz
Reference solvent: Tetrahydrofuran

Test substance formulations in propylene glycol were noted to be stable for at least 4 hours at all concentrations tested. In addition, these test substance preparations formed a homogeneous suspension. The accuracy of dose preparations were within the range of 93% to 96% of nominal, which was considered to represent an acceptable level of accuracy for formulations of this type.

Duration of treatment / exposure:

Test duration: 28 days (followed by a 14-day recovery period)

Frequency of treatment:

Once daily for 28 days, approximately the same time each day, 7 days per week

No. of animals per sex per dose:

Main group 1: 0mg/kg bw/day: 5 male animals and 5 female animals
Recovery group 1: 0mg/kg bw/day: 5 male animals and 5 female animals
Main group 2: 50mg/kg bw/day: 5 male animals and 5 female animals
Main group 3: 200mg/kg bw/day: 5 male animals and 5 female animals
Main group 4: 1000mg/kg bw/day: 5 male animals and 5 female animals
Recovery group 4: 1000mg/kg bw/day: 5 male animals and 5 female animals

Details on study design:

DOSE LEVEL SELECTION:
A 5-day range finding study was performed (with 3 rats/sex/group at dose levels of 50, 200 and 1000 mg/kg/day). The following changes were detected:
Mortality: No mortality.
Clinical signs: No toxicological significant findings.
Body weight and food consumption: Low for females treated at 1000 mg/kg/day only.
Macroscopic examination: No findings.
Liver/Kidney weight: Normal.

Based on these observations, dose levels for a study of 28-days duration were selected to be 0, 50, 200 and 1000 mg/kg/day.

TREATMENT:
- Method: Oral gavage, using a stainless steel stomach tube.
- Frequency: Once daily for 28 days, approximately the same time each day, 7 days per week.
- Pilot study: A pilot study was performed to establish the following dose levels.
- Dose Levels:
Group 1: 0 mg/kg b.w./day (vehicle propylene glycol)
Group 2: 50 mg/kg b.w./day
Group 3: 200 mg/kg b.w./day
Group 4: 1000 mg/kg b.w./day

- Dose volume: 5 ml/kg body weight. Actual dose volumes were calculated weekly according to the latest body weight

Observations and examinations performed and frequency:

- Clinical signs: At least once daily. The time of onset, degree and duration were recorded.
- Mortality / Viability: Twice daily
- Body weights: Weekly and on the day preceding termination, prior to overnight fasting.
- Food consumption: Weekly
- Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected
- Opthalmoscopic examinations: Both eyes were examined following instillation examinations of tropicamide solution (5 nig/ml) during week 4 of treatment (all groups) and week 6 (recovery groups)

Sacrifice and pathology:

NECROPSY:
All animals surviving to the end of the observation period (day 29 for the Main Groups and day 43 for the Recovery Groups) were deeply anaesthetised using ether vapour and subsequently exsanguinated. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded.

Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4%
formaldehyde solution:
Adrenal glands
Aorta
Brain
Cecum
Cervix
Clitoral gland
Colon
Duodenum
Epididymides
Eyes with optic nerve and Harderian gland
Female mammary gland area
Femur including joint
Heart
Ileum
Jejunum
Kidneys
Larynx
Lacrimal gland, exorbital
Liver
Lung, infused with formalin
Lymph nodes - mandibular, ruesenteric
Nasopharynx
Oesophagus
Ovaries
Pancreas
Pituitary gland
Preputial gland
Prostate gland
Rectum
Salivary glands - mandibular, sublingual
Sciatic nerve
Seminal vesicles
Skeletal muscle
Skin
Spinal cord - cervical, midthoracic, lumbar
Spleen
Sternum with bone marrow
Stomach
Testes
Thymus
Thyroid including parathyroid
Tongue
Trachea
Urinary bladder
Uterus
Vagina
All gross lesions

Other examinations:

CLINICAL LABORATORY INVESTIGATIONS:
Blood samples were collected under light ether anaesthesia immediately prior to post mortem examination, between 8.00 and 10.00 a.m.. The animals were fasted overnight before blood sampling, but water was provided. Blood samples were drawn from the retro-orbital sinus of all rats/sex/group and collected into tubes prepared with EDTA for haematological parameters (0.6 ml), tubes prepared with citrate for blood clotting times (1.0 ml) and untreated tubes for clinical biochemistry parameters (>2.0 ml).

Statistics:

The following statistical methods were used to analyse the body weight, food consumption, organ weights and clinical laboratory data:

Univariate one-way analysis of variance was used to assess the significance of intergroup differences.

If the variables could be assumed to follow a normal distribution, the Dunnett-test (many to one t-test) based on a pooled variance estimate was
applied for the comparison of the treated groups and the control groups.

The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.

All tests were two-sided and in all cases p<0.05 was accepted as the lowest level of significance.

Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.

Individual values, means, standard deviations and statistics were rounded off before printing. For example, test statistics were calculated on the
basis of exact values for means and pooled variances and then rounded off to two decimal places. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

The exact Fisher-test was applied to the ophthalmoscopic examination data.

Details on results:

CLINICAL SIGNS AND MORTALITY:
Changes in clinical appearance observed among animals receiving 1000 mg/kg/day included salivation usually accompanied with rales, hunched
posture and rough coat. Salivation and associated rales did not occur immediately after withdrawal of oral treatment. Since these are common
findings following oral gavage of a xenobiotic agent using a stainless steel stomach tube and possibly related to an irritant effect or bad taste
of the test substance, no toxicological significance was attached. In contrast hunched posture was seen in 10/10 females and rough coat in
4/10 females of the high treatment group during, respectively, weeks 3 to 4 and week 4 of treatment, and both findings remained present during the2 weeks of recovery.
No treatment-related mortality occurred during the 6-week study period. On the day of study termination, animal 32 (Control female) was found
dead. No indications of the cause of death were found in this study.

BODY WEIGHT AND WEIGHT GAIN:
Body weights and body weight gain of males receiving 1000 mg/kg/day were lower than controls with statistically significance, during week 2 to 4 of
treatment and during the 2 weeks of recovery. No such decrease was noted among treated females of this group or animals receiving 200 or 50
mg/kg/day

FOOD CONSUMPTION:
There were no differences in food consumption before or after allowance for body weight between treated and control animals during the treatment
and subsequent recovery period

OPHTHALMOSCOPIC EXAMINATION:
There were no ophthalmoscopic findings at weeks 4 and 6 that could be attributed to treatment with the test substance. Incidental findings noted among treated males and females, such as lens opacity, persistent pupillary membrane, and iris malformation, were among those normally seen in rats of this age and strain and considered the result of biological variation

HAEMATOLOGY:
At weeks 4 and 6 of the study, haematological parameters of treated rats were considered not to have been affected by treatment with the test substance. Fluctuations in red blood cell parameters, that were statistically significant in treated animals when compared to controls, showed no
treatment-related distribution or consistency between the sexes. Therefore no toxicological significance was attached to these findings.

CLINICAL BIOCHEMISTRY:
No changes were detected in clinical biochemistry parameters that were considered to have arisen as a result of treatment. At week 4, statistically significant changes were seen in a few clinical biochemistry parameters of animals treated at 1000 mg/kg/day in comparison with corresponding controls. In males an increase was noted in alanine aminotransferase and decrease in total bilirubin, glucose and alkaline phosphatase. Although alanine aminotransferase was the only value that was slightly out of the range of normal background variation, this change was considered not toxicological significant as no comparable changes were detected in the opposite sex or other parameters examined. Statistically significant differences between controls and females receiving 1000 mg/kg/day, in aspartate aminotransferase and potassium, were all within normal limits for rats of this age and strain and considered to have arisen as a result of relatively high or low control values.

Low statistically significant inorganic phosphate values in males of the intermediate groups (50 and 200 mg/kg/day) showed no relationship with
treatment and were considered to have occurred by chance.

At week 6, all statistically significant differences, between animals previously treated with 1000 mg/kg/day and control animals, did not exceed
the range of' normal biological variation for rats of this age and strain and were therefore considered of no toxicological value

ORGAN WEIGHTS:
At week 4, organ weights and relative organ weights (relative to the body weight) of treated animals were indistinguishable from those of control
animals. At week 6, statistically significant absolute or relative (to the body weight) organ weights were present in males and females previously treated at 1000 mg/kg/day in comparison with controls. However, these were considered of no toxicological significance as they remained in
biologically normal limits for rats of this age and strain

GROSS PATHOLOGY:
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment. The incidence and severity of' abnormalities in treated rats where among those commonly seen in rats of this age and strain.

HISTOPATHOLOGY:
Treatment-related changes were detected fn the forestomach upon microscopic examinations. After 4 weeks of treatment, all males and females receiving 1000 mg/kg/day showed hyperplasia of the keratinising epithelium of the forestomach. In a few animals, severe to very severe hyperplasiawas accompanied by moderate to severe submucosal oedema, by distinct necrotic areas and by infiltrates of inflammatory cells. None of these changes were visible in animals receiving 200 or 50 mg/kg/day or after 2 weeks recovery in animals previously treated with 1000 mg/kg/day.

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: original NCD unit is mg/kg/day.

Critical effects observed:

not specified

Conclusions:

Test substance preparations in propylene glycol appeared to be stable and homogeneous. Moreover, sufficiently accurate concentrations were
encountered for the purpose of this study.

Toxicological significant changes were detected in animals treated at 1000 mg/kg/day only. The first signs appeared in males and females during the third week of treatment and consisted of hunched posture and rough coat. In addition, males showed also lower body weights. All these changes continued during the fourth week of treatment and the 2-week recovery period.

Males and females of the 1000 mg/kg group showed hyperplasia of the keratinising epithelium of the forestomach upon microscopic examination.
In a few animals this was accompanied with submucosal oedema, distinct necrotic areas and infiltration of inflammatory cells. These findings indicatea direct local toxic effect of the test substance to the stomach following oral treatment with a stomach tube, and may be indirectly related to the decrease in physical condition of the animals as noted above.

There were no changes in food consumption, ophthalmoscopic examination, clinical laboratory investigations, macroscopic examination and organ weights that were considered to be an effect of treatment.

From these results a definitive No Observed Effect Level (NOEL) of 200 mg/kg/day was established.

Executive summary:

In a GLP compliant, guideline sub-acute oral toxicity test the test substance was administered to male and female rats by daily oral gavage for a period of 28 days (followed by a 14 -day recovery period) at dose levels of 0 mg/kg bw/day (vehicle propylene glycol) 50 mg/kg bw/day, 200 mg/kg bw/day and 1000 mg/kg bw/day.

Toxicological significant changes were detected in animals treated at 1000 mg/kg/day only. The first signs appeared in males and females during the third week of treatment and consisted of hunched posture and rough coat. In addition, males showed also lower body weights. All these changes continued during the fourth week of treatment and the 2-week recovery period. Males and females of the 1000 mg/kg group showed hyperplasia of the keratinising epithelium of the forestomach upon microscopic examination. In a few animals this was accompanied with submucosal oedema, distinct necrotic areas and infiltration of inflammatory cells. These findings indicate a direct local toxic effect of the test substance to the stomach following oral treatment with a stomach tube, and may be indirectly related to the decrease in physical condition of the animals as noted above. There were no changes in food consumption, ophthalmoscopic examination, clinical laboratory investigations, macroscopic examination and organ weights that were considered to be an effect of treatment. From these results a definitive No Observed Effect Level (NOEL) of 200 mg/kg/day was established

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP compliant guideline sub-acute study with a klimisch score of 1

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a GLP compliant, OECD test guideline 407 sub-acute oral toxicity test the test substance was administered to male and female rats by daily oral gavage for a period of 28 days (followed by a 14 -day recovery period) at dose levels of 0 mg/kg bw/day (vehicle propylene glycol) 50 mg/kg bw/day, 200 mg/kg bw/day and 1000 mg/kg bw/day.

Toxicological significant changes were detected in animals treated at 1000 mg/kg/day only. The first signs appeared in males and females during the third week of treatment and consisted of hunched posture and rough coat. In addition, males showed also lower body weights. All these changes continued during the fourth week of treatment and the 2-week recovery period. Males and females of the 1000 mg/kg group showed hyperplasia of the keratinising epithelium of the forestomach upon microscopic examination. In a few animals this was accompanied with submucosal oedema, distinct necrotic areas and infiltration of inflammatory cells. These findings indicate a direct local toxic effect of the test substance to the stomach following oral treatment with a stomach tube, and may be indirectly related to the decrease in physical condition of the animals as noted above. There were no changes in food consumption, ophthalmoscopic examination, clinical laboratory investigations, macroscopic examination and organ weights that were considered to be an effect of treatment. From these results a definitive No Observed Effect Level (NOEL) of 200 mg/kg/day was established

Justification for classification of repeated dose toxicity via oral route-systemic effects endpoint:

GLP compliant guideline study with a klimisch score of 1