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Administrative data

Description of key information

A 7 day repeated dose toxicity range finding study is available. At 1000 mg/kg bw  animals had to be killed due to excessive signs to treatment on day 3 of treatment. Effects seen were underactive behaviour, shallow breathing and unsteady/prostrate posture. Animals also showed body weight loss and low food consumption. At 400 mg/kg bw animals showed underactivity and low food consumption. At 150 mg/kg bw  no clinical signs, body weight effects or macroscoping findings were seen. A subsequent 28-day study was not (yet) performed. 

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:
supporting study
Study period:
between November 2006 and February 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: 7-Day Range-Finding Oral (Gavage) Toxicity Study was done to standard test method by a reliable GLP laboratory.
Qualifier:
no guideline required
Guideline:
other: 7-Day Range-Finding Oral (Gavage) Toxicity Study in the Wistar Rat
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
A total of ten male and ten female Crl:CD® (SD)IGS BR rats were received from Charles River (UK) Ltd,. The rats were ordered at 29 to 35 days of age and within a weight range of 125 to 153g for males and 112 to 129g for females.

On arrival, the animals were removed from the transit boxes and allocated to study cages. Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals. Each sex was allocated separately.

The cages constituting each group were blocked together by sex and the groups were dispersed in batteries so that possible environmental influences arising from their spatial distribution were equilibrated, as far as was practicable. Additionally, batteries of cages were rotated around the room at weekly intervals to further minimise possible spatial variations.

Each animal was assigned a number and identified uniquely within the study by a tail tattoo. Each cage label was colour-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupants.

Before the start of treatment, the animals were allowed to acclimatise to the conditions described below for six days before treatment commenced. For those animals selected for this study, their age at the start of treatment was 35 to 41 days and their bodyweights were in the range of 168 to 200 g for males and 150 to 167 g for females.

Animals were housed inside a barriered rodent facility (Building 8, Room 19). The facility was designed and operated to minimise the entry of external biological and chemical agents and to minimise the transference of such agents between rooms. Before the study the room was cleaned and disinfected.

Each animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated. The temperature and relative humidity controls were maintained within the range of 19 to 23°C and 40 to 70% respectively. Artificial lighting was controlled to give a cycle of 12 hours continuous light and 12 hours continuous dark per 24 hours.

Periodic checks were made on the number of air changes in the animal rooms. Temperature and humidity were monitored continuously and recorded daily. Since these data show that there were no significant deviations from target values they are not presented.

The animals were housed three of one sex per cage unless this number was reduced by mortality or isolation. The cages were made of a polycarbonate body and floor with a stainless steel mesh lid. The cages had wood shavings as bedding (lignoceI 3-4 wood flakes). Cages, bedding, food hoppers and water bottles were changed at appropriate intervals.

The animals were allowed free access to a standard rodent diet (Rat and Mouse No. 1 Maintenance Diet), except when urine was being collected and overnight before routine blood sampling. This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.

Potable water taken from the public supply was freely available via polycarbonate bottles fitted with sipper tubes. Each animal was provided with an Aspen chew block for environmental enrichment. Chew blocks were provided throughout the study and were replaced when necessary. Certificates of analysis were received routinely from the water supplier. Certificates of analysis were received routinely from the supplier of the aspen chew blocks. Since the results of these various analyses did not provide evidence of contamination that might have prejudiced the study, they are not presented.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:

VEHICLE
The test substance, Montaverdi, was prepared for administration as a series of graded concentrations in the vehicle, by adding small amounts of vehicle to the test material and mixing using a spatula. This mixture was then made up to the required volume, using the vehicle, and ensured to be homogenous by mixing using a high sheer mixer. The test substance was used as supplied. All formulations were prepared freshly each day and used within 48 hours of preparation.

Group Treatment Dosage Concentration Volume dosage
mg/kg/day) (mg/mL) (mL/kg)
2 Montaverdi 150 30 5
3 Montaverdi 400 80 5
4 Montaverdi 1000 200 5
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
7 days.
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
150, 400 or 1000 mg/kg body weight
Basis:
actual ingested
No. of animals per sex per dose:
The study consisted of three treated groups of rats, identified as follows:
Group Treatment Dosage# No. of animals Animal numbers Cage numbers
(mg/kg/day) Male Female Male Female Male Female
1 Montaverdi 150 3 3 1 -3 10-12 1 4
2 Montaverdi 400 3 3 4-6 13-15 2 5
3 Montaverdi 1000 3 3 7-9 16-18 3 6

Control animals:
yes, concurrent vehicle
Details on study design:
Administration:
Animals received the test substance or vehicle control formulations orally at a volume-dosage of 5 mL/kg bodyweight, using a suitably graduated syringe and a rubber catheter inserted via the mouth into the stomach.

All animals were dosed in sequence of cage-number within each group, once each day at approximately the same time each day, seven days per week. The volume administered to each animal was calculated from the most recently recorded scheduled bodyweight.

A daily record of the weight of each formulation dispensed and the amount remaining after dosing was made. The balance of these two weights was compared with the predicted usage as a check that the doses had been administered correctly. No significant discrepancy was found.

Observations and examinations performed and frequency:
Activities and Observations
The following observations were recorded:

Clinical observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of ill-health amongst the occupant(s). Any deviation from nonnal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.


Mortality
Debilitated animals were observed carefully and animals judged in extremis were killed. Animals were also killed to prevent unnecessary or prolonged suffering. A complete necropsy was perfonned in all cases as described below.

Bodyweight
The weight of each rat was recorded before treatment commenced (Day -4), on the day that treatment commenced (Day 0), once during the treatment period and before necropsy.

Food consumption
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for each cage during the treatment period. From these records the mean weekly consumption per animal (glratlweek) was calculated for each cage.

Water consumption
Daily visual water consumption was assessed each day of treatment.

NECROPSY AND HISTOLOGY
Method of kill: Animals killed during the study and those surviving until the end of the scheduled treatment period were killed by carbon dioxide asphyxiation. The sequence in which the animals were killed after completion of treatment was selected to allow satisfactory inter-group comparison.

Macroscopic pathology:
All animals were subject to a detailed necropsy.
After a review of the history of each animal, a full macroscopic examination of the tissues was performed. The cranial roof
was removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position, morphology or interaction was recorded.

Organ weights
The following organs, taken from each animal killed after seven days of treatment, were
dissected free of adjacent fat and other contiguous tissue and the weights recorded:
Liver
Kidneys
Spleen

Fixation
Macroscopically abnormal tissues were fixed and retained. These included livers, kidneys and uterus with cervix.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
animals had to be terminated for the 1000 mg/kg dose range, please see table 1 in the attachments section.
Mortality:
mortality observed, treatment-related
Description (incidence):
animals had to be terminated for the 1000 mg/kg dose range, please see table 1 in the attachments section.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
as expected, but low for the 1000 mg/kg dose range
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
results very low for animals dosed at 1000 mg/kg
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Mortality:
There were no unscheduled deaths in animals receiving 150 or 400 mg/kg/day.
On Day 3 of treatment all animals receiving 1000 mg/kg/day were killed due to the excessive signs of reaction to treatment. Signs of reaction to treatment included underactive behaviour (in all animals from the completion of dosing), shallow breathing, piloerect coat, partially closed eyes and unsteady/prostrate posture. The combination of these findings resulted in animals being moribund and animals were therefore sacrificed prematurely. As documented, premature sacrifice of animals given 1000 mg/kg/day resulted in full organ weights not being recorded for these animals.

Clinical signs: Please see table 1 in the attached background material section.
No signs of reaction to treatment were seen in animals receiving 150 mg/kg/day. Signs of reaction to treatment in animals receiving 400 mg/kg/day were restricted to salivation in two males and two females on completion of dosing the group on Day 6 of treatment and underactive behaviour in all males 1 to 2 hours after dosing on Day 7 of treatment.

On Day 2 all animals receiving 1000 mg/kg/day showed post-dose signs but not until the last check of the day (approximately 6 hours after dosing). These signs included piloerection, underactivity, shallow breathing and partially closed eyelids. Although present at the end of the working day they had resolved in all animals prior to dosing the following day. On Day 3 underactivity was apparent in all animals by the end of dosing the group and at the 1-2 hour check, other signs, some consistent with those seen the previous day (see Mortality section above), were also present. On Day 3, not only was the degree of the signs more severe than the previous day but also the onset after dosing was sooner, which indicates possible slow clearance of Montaverdi.

Bodyweight:
Bodyweight gains were as anticipated for animals receiving 150 or 400 mg/kg/day but were low for animals receiving 1000 mg/kg/day.

Food consumption:
Food consumption was as anticipated for animals receiving 150 mg/kg/day but was slightly low for animals receiving 400 mg/kg/day. As animals receiving 1000 mg/kg/day were killed on Day 3 of treatment data has been calculated for this three day period and food consumption was markedly low for these animals.

Water consumption
There was no visual effect on water consumption for any animals during the treatment period.

Organ weights
Spleen weights for animals given 150 or 400 mg/kg/day were generally as expected, although mean spleen weights for both sexes given 400 mg/kg/day were lower than those recorded in those given 150 mg/kg/day.

Similarly, kidney and liver weights for animals given 150 or 400 mg/kg/day were as expected, but there was a tendency for liver weights for males given 400 mg/kg/day to be slightly lower than those given 150 mg/kg/day.

Macropathology:
Animals given 150 or 400 mg/kg/day showed no macroscopic abnormalities after 7 days of treatment.
Dose descriptor:
NOEL
Effect level:
> 150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects seen at this dose range.
Critical effects observed:
not specified

The daily oral (gavage) administration of Montaverdi, a fragrance ingredient used in household products such as fabric conditioners, to Cr1:CD® (SD)IGS BR rats at dosages up to 400 mg/kg/day was tolerated with no death; treatment at 1000 mg/kg/day was not tolerated with the death of all animals.

Although over the 7-day treatment period a dosage of 400 mg/kg/day was tolerated the presence of post-dose underactivity (a sign also seen at 1000 mg/kg/day which contributed to the early termination of that group) in all males on the last day of treatment, is an indication that over a longer period of dosing (such as 28 days) 400 mg/kg/day may not be tolerated. On this basis either an additional preliminary study should be performed to assess a suitable level for a 28-day study or the high level for such a study should be closer to 150 mg/kg/day.

If an additional study was performed, a suitable design would be: a single sex (males would be recommended, as this sex receiving 400 mg/kg/day showed post dosing signs prior to termination), up to three treatment levels (possible dose levels could be 150, 300 and 400 mg/kg/day) and a longer duration of treatment up to 14 days, this would enable further assessment of the potential for a delayed response.

In conclusion, based on data from this 7 day repeat dose toxicity study a suitable high level for a 28-day toxicity study should be below 400 mg/kg/day, a level closer to 150 mg/kg/day than 400 mg/kg/day may be suitable.

Conclusions:
In conclusion, based on data from this 7 day repeat dose toxicity study a suitable high level for a subsequent 28-day toxicity study (Huntingdon Life Sciences Study Number IFF/0336) should be below 400 mg/kg/day, a level closer to 150 mg/kg/day than 400 mg/kg/day may be suitable.
Executive summary:

The systemic toxic potential of Montaverdi (a fragrance ingredient used in household products such as fabric conditioners), to Crl:cn® (SD)IGS BR rats by oral administration was assessed over a period of seven days. Three groups, each comprising three male and three female rats received Montaverdi at dosages of 150, 400 or 1000 mg/kg/day. On Day 3 of treatment all animals receiving 1000 mg/kg/day were killed due to the excessive signs of reaction to treatment. Signs of reaction to treatment included underactive behaviour, shallow breathing, piloerect coat, partially closed eyes and unsteady/prostrate posture, these signs did not show any evidence of resolution and approximately 3 hours after dosing animals were found to have little or no righting or pinch reflex. In addition the animals from this treatment-level were showing bodyweight loss and low food consumption. Macroscopic examination revealed small thymus and spleen, pale thymus, kidneys and liver and changes in the stomach. Signs of reaction to treatment in animals receiving 400 mg/kg/day were confined to post-dose underactivity in all males on the last day of treatment (Day 7) and post-dose salivation on a single occasion for 2 animals of each sex. Signs of reaction to treatment in animals receiving 1000 mg/kg/day started on Day 1 with most animals showing a piloerect coat approximately 1 to 2 hours after dosing. On Day 2 post-dosing signs were seen at the end of the working day, they included piloerection, underactivity, shallow breathing and partially closed eyelids. On Day 3 of treatment these signs although not present prior to dosing became apparent 1-2 hours after dosing and were considered more severe in nature (including no righting reflex and unconsciousness). Signs showed no resolution and at approximately 5 hours after dosing animals were moribund, necessitating premature sacrifice. Bodyweight gains were as anticipated for animals receiving 150 or 400 mg/kg/day but were low for animals receiving 1000 mg/kg/day, with all animals receiving 1000 mg/kg/day showing a loss of at least 20 g. Food consumption was as anticipated for animals receiving 150 mg/kg/day but was slightly low for animals receiving 400 mg/kg/day and markedly low for animals receiving 1000 mg/kg/day. There was no visual effect on water consumption for any animals during the treatment period. There were no marked changes in organ weights observed in animals given 150 or 400 mg/kg/day, however spleen weights were lower for all animals given 1000 mg/kg/day. Animals given 150 or 400 mg/kg/day showed no macroscopic abnormalities after 7 days of treatment. In conclusion, based on data from this 7 day repeat dose toxicity study a suitable high level for a subsequent 28-day toxicity study (Huntingdon Life Sciences Study Number IFF/0336) should be below 400 mg/kg/day, a level closer to 150 mg/kg/day than 400 mg/kg/day may be suitable.

Endpoint conclusion
Endpoint conclusion:
no study available

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

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
A 7 day range finding study is available which is included for completeness

Justification for classification or non-classification