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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9 August - 14 September 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Guideline:
other: OECD Guideline 424 (Neurotoxicity study in Rodents)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Product name: Antiblaze V6
Appearance: clear pale yellow liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, France
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: 197 - 231 g (males), 142 - 181 (females)
- Housing: individual, in wire-mesh cages
- Diet: ad libitum (A04 C pelleted maintenance diet)
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): approximately 12
- Photoperiod (hrs dark / hrs light): 12h/12h

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
VEHICLE: olive oil
- Concentration in vehicle: 3, 30 or 120 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day
- Lot/batch no. (if required): Cooper (Melum France) A29902/3
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Dose levels based on results of a 7-day range-finding toxicity study (oral: gavage, rat)
- Concentration of each dosage determined on day 1. Results demonstrated satisfactory homogeneity and acceptable correspondence between nominal and measured concentrations (in range of +-12%)
Duration of treatment / exposure:
29 days
Frequency of treatment:
once a day, at approximately the same time each day, 7 days per week for 29 days.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
15 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
150 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
600 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
5 males and 5 females per dose level
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: morbidity and mortality at least twice a day during the treatment period, general clinical signs: once per day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before beginning of treatment and once a week after

BODY WEIGHT: Yes
- Time schedule for examinations: once before allocation to groups, on first day of treatment and then once a week until end of the study

FOOD CONSUMPTION: Yes
- quantity of food consumed by the animals of each cage recorded once a week until end of the study

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before daily treatment, at the end of the treatment period
- Animals fasted: Yes (overnight)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at end of the treatment period

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at end of treatment period
- Dose groups that were examined: all
- Battery of functions tested: reactivity to manipulation and stimuli / motor activity
Sacrifice and pathology:
- Sacrifice on completion of the treatment period, after at least 14 hours fasting
- Organ weights were recorded as soon as possible after dissection of Adrenals, brain, epididymydes, heart, kifneys, liver, ovaries, spleen, testes, thymus, thyroids with parathyroids.
- Complete macroscopic post-mortem examination on all study animals
- Histopathology was performed in the control and high dose group on all organs weighed and the follwing tissues: Cecum, Colon, Duodenum, Ileum, Jenunum, lungs with bronchi, mandibular and mesenteric lymph nodes, prostate, sciatic nerve, seminal vesicles, spinal cord, sternum with bone marrow, stomach with forestomach, urinary bladder, uterus, vagina
On all macroscopic lesions in all dose groups, on the liver of females in the mid dose group.
Statistics:
Dunn test for analysis of body weight, food consumption, hematology, blood biochemistry and organ weight data.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality, no treatment related clincal signs were observed.

BODY WEIGHT AND WEIGHT GAIN
A slight decrease in weight gain was seen in males (–16% in high-dose group) and a slightly increased weight gain in females (+9% in high-dose group). The changes were not statistically significant, not dose related and not consistent between sexes and are therefore not considered treatment related.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No treatment related effects were observed.

HAEMATOLOGY
A statistically significant higher mean prothrombin time (21.5s Vs 15s; 42%, p<0.01) was seen in high dose males. However, all values remained within the historical background data of the test laboratory. In addition, the effect was not observed in treated female animals. Consequently, the changes were not considered to be of toxicological significance. There were no other treatment-related effects on haematological parameters.

CLINICAL CHEMISTRY
A significantly higher mean cholesterol level was detected in the high dose males (+43%) and females (+63%). These increases were greater than the historical control values. Lower mean alkaline phosphatase values at 600 mg/kg/day in rats were significant (-44% in males and –32% in females) but within the historical control range and not considered to be toxicologically relevant.

URINALYSIS
not performed.


NEUROBEHAVIOUR
No treatment-related alterations in autonomic or physiological functions were observed and there were no changes in neurotoxicological parameters. A slight to moderate dose-dependent increase in motor activity was seen in treated male animals. The increases were 4%, 11% and 19% greater than controls in treated males at 15, 150 and 600 mg/kg/day, respectively. In females, total motor activity was also increased, although not in a dose-dependent manner. The increases were 34%, 23% and 28% in treated females at 15, 150 and 600 mg/kg/day, respectively. In the absence of other changes in the functional observation battery these findings are not considered of toxicological significance.

ORGAN WEIGHTS
Significantly greater absolute (35% and 78%) and relative (30% and 73%) liver weight was noted in females at 150 mg/kg/day and 600 mg/kg/day respectively and in males at 600 mg/kg/day (absolute: 52% and relative: 68%). A biologically significant increase in absolute and relative thyroid weight was also noted in rats dosed with 600 mg/kg/day. The absolute weights were increased by 36 and 58% and the relative weights were increased by 48 and 54% in males and females, respectively, when compared to controls.

GROSS PATHOLOGY
Macroscopic examination showed liver enlargement in 9/10 animals in the high dose group. No other treatment related changes were observed.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopic evaluation revealed treatment related changes only in the liver and thyroid.
A slight to marked hepatocellualr hypertrophy was observed in all animals of the 600 mg/kg/day dose group. However, there was no evidence of nuclear or cytoplasmic degenerative or necrotic changes. This correlates well with the absence of any transferase activity changes in the blood biochemistry examination.
A slight hepatocelluar hypertrophy was observed in 3 of 5 female rats at 150 mg/kg bw.
Thyroid microscopy revealed follicular cell hypertrophy, decreased diameter of follicular lumen and decreased eosinophilic colloidal contents in all animals of the high dose group. These changes are indicative of an increased thyroid activity. This is frequently observed after liver enzyme induction due to increase thyroid hormone turnover in the liver and can thus be secondary to the liver hypertrophy.
The effects in the mid dose, an increases in liver weight in females combined with a slight hepatocelluar hypertrophy can also be regarded as adaptive changes due to liver enzyme induction. Therefore the NOAEL is considered conservative.






Effect levels

Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The No Adverse Observed Effect Level (NOAEL) of the test material, in the Sprague-Dawley rat was found to be 15 mg/kg bw/day based on liver weight increase and slight hepatocellular hypertrophy in female animals of the 150 mg/kg dose group. As the effects in the mid dose can also be regarded as adaptive changes due to liver enzyme induction, rather than adverse, the NOAEL is considered conservative.
Executive summary:

In short-term oral repeated dose toxicity study, 2,2-bis(chloromethyl)trimethylene bis(bis(2-chloroethyl)phosphate) was administered to 20 male and 20 female Sprague-Dawley rats by gavage at dose levels of 0, 15, 150 and 600 mg/kg bw/day.

There were no compound related effects in mortality, clinical signs, body weight, food consumption and hematology. At 150 mg/kg/day significantly increased liver weight and slight centrilobular hepatocellular hypertrophy was noted among female animals. At 600 mg/kg/day significant liver weight increase, liver enlargement, centrilobular hepatocellular hypertrophy without evidence of nuclear or cytoplasmic degenerative or necrotic changes, and morphological changes indicative of thyroid hyperactivity as well as an increase in blood cholesterol levels were noted in male and female animals. As the effects in the mid dose can also be regarded as adaptive changes due to liver enzyme induction, rather than adverse, the NOAEL is considered conservative.

The NOAEL under the experimental conditions of this study is 15 mg/kg/day.

 

This repeated dose study in the rat is acceptable and satisfies the guideline requirement for a 28 -day repeated dose oral toxicity study in rodents, according to OECD Guideline No.407.