Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
sub-chronic toxicity: oral
Remarks:
Oral (Dietary) Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 July 2014 - 23 June 2016
Reliability:
1 (reliable without restriction)
Cross-reference
Reason / purpose for cross-reference:
reference to other study
Reference
Endpoint:
reproductive toxicity, other
Remarks:
Oral (Dietary) Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 July 2014 - 23 June 2016
Reliability:
1 (reliable without restriction)
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): Isobutavan
- Physical state: colourless to pale yellow liquid
- Lot/batch No.: SC00011161
- Expiration date of the lot/batch: 08 May 2016
- Storage condition of test material: Sealed container, 2 to 8°C in the dark
Species:
rat
Strain:
other: Crl:WI(Han) strain
Details on species / strain selection:
Rats are obtained from Charles River Laboratories, Margate, UK. The rat was selected because it is a readily available rodent species acceptable to the regulatory authorities and recommended for reproduction studies because of its reproductive characteristics.
Sex:
male/female
Details on test animals or test system and environmental conditions:
At the start of dosing, males weighed between 289.5 and 376.9 g, females weighed between 177.4 and 221.7 g and all animals were 10 to 12 weeks old. The animals were housed in groups of up to two (pre-pairing and post-pairing), one female with one male (pairing) or singly (mated females).
Route of administration:
oral: feed
Type of inhalation exposure (if applicable):
not specified
Vehicle:
unchanged (no vehicle)
Details on exposure:
The dietary route of administration was chosen because it is an acceptable and commonly used route of exposure for regulatory studies of this type.
Details on mating procedure:
During the pairing procedure one male was housed with one female of the same treatment group for up to 15 days. Females which had not shown evidence of mating by Day 10 of the pairing period were paired with proven males of the same treatment group for up to five days. Mating was confirmed by the presence of a vaginal plug in situ or sperm in a vaginal washing. On confirmation of mating, vaginal washing was discontinued and the male was removed. The day on which mating was confirmed was designated Day 0 of gestation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulations were prepared at 300 and 25000 mg Isobutavan/kg diet and analysed for stability and homogeneity. Samples for homogeneity analysis were also taken from formulations prepared for use on the first and last days of dosing. Samples for analysis of achieved concentration were taken from formulations prepared for use on the first and last days of dosing. Samples were analysed by the Formulations Analysis section of the Central Dispensary at Covance.
Duration of treatment / exposure:
The test article was administered, ad libitum, in the diet to males for two weeks prior to pairing, during the pairing period and a further two weeks before necropsy. The males were provided with diet containing test article for a minimum of six weeks prior to necropsy.
The test article was administered, ad libitum, in the diet to females for two weeks prior to pairing, during pairing and until Day 4 post-partum, inclusive. The females were allowed to litter and rear their offspring to Day 4 post-partum
Frequency of treatment:
ad libitum
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 "Control" : 10 males and 10 females
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 2 "Low" : 10 males and 10 females
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Group 3 "Intermediate" : 10 males and 10 females
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group 4 "High" : 10 males and 10 females
No. of animals per sex per dose:
1 dose concentration per group of 10 males and 10 females
Control animals:
yes, concurrent no treatment
Details on study design:
Dose levels were based on the absence of findings at the limit dose in a 14-day dietaryrange-finding study. The high dose was selected to indicate toxicity or its absence at the limit dose. The intermediate dose was based on a standard progression increase from the low dose. The low dose was chosen to approximate a No Observed Adverse Effect Level (NOAEL) value for a similar substance.
Positive control:
No
Parental animals: Observations and examinations:
The following were assessed: clinical observation, body weight, food intake, mating, litter data, functional observations and tests, locomotor activity, haematology, clinical chemistry, organ weights, gross pathology and microscopic pathology.
Oestrous cyclicity (parental animals):
Yes
Sperm parameters (parental animals):
Yes
Litter observations:
Yes
Postmortem examinations (parental animals):
Yes
Postmortem examinations (offspring):
Yes
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: NOAEL for males and females was 1000 mg/kg bw/day.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
no
Dose response relationship:
no
Relevant for humans:
no
Clinical signs:
no effects observed
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: NOAEL for males and females was 1000 mg/kg bw/day
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
no
Dose response relationship:
no
Relevant for humans:
no
Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
no
Dose response relationship:
no
Relevant for humans:
no

In conclusion, Isobutavan administered in the diet to male and female rats at 100, 300 or 1000 mg Isobutavan/kg bodyweight/day elicited no adverse effects. Under the conditions of the current study, the no-observed-adverse-effect-level (NOAEL) for adult toxicity and reproductive and developmental toxicity was established as nominal 1000 mg Isobutavan/kg body weight/day (achieved mean high dose level of 986.4 mg/kg/day for males and 1215.3 mg/kg/day for females).

Conclusions:
In conclusion, Isobutavan administered in the diet to male and female rats at 100, 300 or 1000 mg Isobutavan/kg bodyweight/day elicited no adverse effects.

Under the conditions of the current study, the no-observed-adverse-effect-level (NOAEL) for adult toxicity and reproductive and developmental toxicity was established as nominal 1000 mg Isobutavan/kg body weight/day (achieved mean high dose level of 986.4 mg/kg/day for males and 1215.3 mg/kg/day for females).
Executive summary:

The study was performed in accordance to the OECD 422 guidelines (Adopted 22 March 1996), which were current at the time this study was conducted. The dietary route of administration was chosen because it is an acceptable and commonly used route of exposure for regulatory studies of this type.

The test article was administered, ad libitum, in the diet to males for two weeks prior to pairing, during the pairing period and a further two weeks before necropsy. The males were provided with diet containing test article for a minimum of six weeks prior to necropsy. The test article was administered, ad libitum, in the diet to females for two weeks prior to pairing, during pairing and until Day 4 post-partum, inclusive. The females were allowed to litter and rear their offspring to Day 4 post-partum. The following were assessed: clinical observation, body weight, food intake, mating, litter data, functional observations and tests, locomotor activity, haematology, clinical chemistry, organ weights, gross pathology and microscopic pathology.

Diet formulations were considered acceptable for use. The mean results were within the range of 101 to 109% of nominal (target range 85 to 110% of nominal). Test article consumption was within 20% of the target dose with only a few exceptions, all of which involved consumptions greater than the target dose. All animals survived until their scheduled kill. There were no clinical observations that were considered to be related to the test article.

All animals gained weight over the duration of the study. Although there were some fluctuations and differences between groups in body weight gain, there was no clear dose relationship and these were considered most likely due to palatability of the diet containing Isobutavan and not indicative of toxicity. There was a dose-dependent decrease in food consumption in males across all dose levels between Days 1 and 4 of the pre-pairing phase compared to the control males. Males offered 1000 mg Isobutavan/kg body weight/day continued to consume less diet than the control males through the rest of the pre-pairing phase and throughout the post-pairing phase. There was no similar pattern for females during this phase. There was no effect on clinical pathology and no effect in the neuro-behavioural tests conducted. An assessment of the reproductive function revealed no adverse effects. Qualitative assessment of the testis did not indicate any abnormalities in the integrity of the various cell types present within the different stages of the spermatogenic cycle. There was no effect of Isobutavan on organ weights. Macroscopically, tissues were unremarkable and there were no microscopic findings indicative of Isobutavan-related effects. An assessment of the development of the first generation to Day 4 post-partum revealed no adverse effects.

In conclusion, Isobutavan administered in the diet to male and female rats at 100, 300 or 1000 mg Isobutavan/kg bodyweight/day elicited no adverse effects. Under the conditions of the current study, the no-observed-adverse-effect-level (NOAEL) for adult toxicity and reproductive and developmental toxicity was established as nominal 1000 mg Isobutavan/kg body weight/day (achieved mean high dose level of 986.4 mg/kg/day for males and 1215.3 mg/kg/day for females).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
4-formyl-2-methoxyphenyl isobutyrate
EC Number:
243-956-6
EC Name:
4-formyl-2-methoxyphenyl isobutyrate
Cas Number:
20665-85-4
Molecular formula:
C12H14O4
IUPAC Name:
4-formyl-2-methoxyphenyl 2-methylpropanoate
Test material form:
other: liquid
Specific details on test material used for the study:
- Name of test material (as cited in study report): Isobutavan
- Physical state: colourless to pale yellow liquid
- Lot/batch No.: SC00011161
- Expiration date of the lot/batch: 08 May 2016
- Storage condition of test material: Sealed container, 2 to 8°C in the dark

Test animals

Species:
rat
Strain:
other: Crl:WI(Han) strain
Details on species / strain selection:
Rats are obtained from Charles River Laboratories, Margate, UK. The rat was selected because it is a readily available rodent species acceptable to the regulatory authorities and recommended for reproduction studies because of its reproductive characteristics.
Sex:
male/female
Details on test animals or test system and environmental conditions:
At the start of dosing, males weighed between 289.5 and 376.9 g, females weighed between 177.4 and 221.7 g and all animals were 10 to 12 weeks old. The animals were housed in groups of up to two (pre-pairing and post-pairing), one female with one male (pairing) or singly (mated females).

Administration / exposure

Route of administration:
oral: feed
Details on route of administration:
The dietary route of administration was chosen because it is an acceptable and commonly used route of exposure for regulatory studies of this type.
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
Formulations were prepared weekly (twice a week) . The formulations were stored frozen in a sealed container, protected from the light.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulations were prepared at 300 and 25000 mg Isobutavan/kg diet and analysed for stability and homogeneity. Samples for homogeneity analysis were also taken from formulations prepared for use on the first and last days of dosing. Samples for analysis of achieved concentration were taken from formulations prepared for use on the first and last days of dosing. Samples were analysed by the Formulations Analysis section of the Central Dispensary at Covance.
Duration of treatment / exposure:
The test article was administered, ad libitum, in the diet to males for two weeks prior to pairing, during the pairing period and a further two weeks before necropsy. The males were provided with diet containing test article for a minimum of six weeks prior to necropsy.
The test article was administered, ad libitum, in the diet to females for two weeks prior to pairing, during pairing and until Day 4 post-partum, inclusive. The females were allowed to litter and rear their offspring to Day 4 post-partum
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 "Control" : 10 males and 10 females
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 2 "low" : 10 males and 10 females
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Group 3 "Intermediate" : 10 males and 10 females
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group 4 "High" : 10 males and 10 females
No. of animals per sex per dose:
1 dose concentration per group of 10 males and 10 females
Control animals:
yes, concurrent no treatment
Details on study design:
Dose levels were based on the absence of findings at the limit dose in a 14-day dietaryrange-finding study. The high dose was selected to indicate toxicity or its absence at the limit dose. The intermediate dose was based on a standard progression increase from the low dose. The low dose was chosen to approximate a No Observed Adverse Effect Level (NOAEL) value for a similar substance.

Examinations

Observations and examinations performed and frequency:
The following were assessed: clinical observation, body weight, food intake, mating, litter data, functional observations and tests, locomotor activity, haematology, clinical chemistry, organ weights, gross pathology and microscopic pathology.

Health Monitoring :
All animals were observed at the beginning and the end of the working day for signs of ill health or overt toxicity.

Clinical Examinations:
Each animal was given a detailed physical examination at weekly intervals. An individual record was maintained of the clinical condition of each animal. The animals were also observed once daily. Observations were not recorded from Day 20 of gestation to Day 4 of lactation (inclusive).

Body Weights:
Body weights were recorded weekly for males, from Day -7 (Pre-dose Day 1) and including the day of, prior to, necropsy. Body weights for females were recorded weekly from Day -7 (Pre-dose Day 1), prior to pairing, and until confirmation of mating. Body weights were then recorded on Days 0, 7, 14 and 20 of gestation and Days 0, 1 and 4 of lactation. Body weights recorded during the pairing period and Day 0 of lactation were for welfare reasons only and are not reported.

Food Consumption:
Food consumption was recorded daily during pre-pairing, pairing and post-pairing (including gestation and lactation phases for females). Food consumption was calculated as g/animal/day. Food consumption recorded during the pairing period was for welfare reasons only and is not reported

Lactation phase:
Animals were observed three times in each working day at the beginning, middle and end of the day starting when the first females reached Day 21 of gestation and until the last female had littered or until a potential Day 25 of gestation, whichever was soonest. The females were observed for signs of the start of parturition for example blood in the cage. The time and date of this observation was recorded where possible and marked the end of gestation.

Functional observational battery:
All animals were subjected to a battery of behavioural tests and observations before initiation of treatment and at once weekly intervals thereafter.

During Post-Paring Week 3 (males) or Lactation Day 4 (females), an assessment was made of sensory reactivity to stimuli, grip strength and motor activity for five animals per sex per group. These were the five males with the highest identification numbers and the five first littered females. At the time of testing, the observer was unaware of each animal’s dose level (‘blinded’ test procedure). Where possible, the observations were performed at the same time on each occasion.
Other examinations:
Pups: A macroscopic examination was performed on pups.
Adult Necropsies, Organ Weights and Macroscopic Observations: All adult animals were subjected to necropsy
Histology: Tissue trimming, processing and embedding was performed for the five adult animals with the highest identification numbers per sex per group for Groups 1 and 4.
Microscopic Observations: Microscopic observations were performed for the five adult animals with the highest identification numbers per sex per group for Groups 1 and 4.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
One female given 100 mg Isobutavan/kg body weight/day had a hunched appearance on Day 8 of the pre-pairing phase, Day 1 of the pairing phase and Day 0 and 7 of gestation. This single observation, in the low dose treatment group was considered not to be related to the test article. Other observations seen were transient and typical of group-housed rats of this age and strain in this laboratory.
Mortality:
no mortality observed
Description (incidence):
All animals survived until their scheduled kill.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
All animals gained weight over the duration of the study. Although there were some fluctuations and differences between groups in body weight gain, there was no clear dose relationship and these were considered most likely due to palatability of the diet containing Isobutavan and not indicative of toxicity.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There was a dose-dependent decrease in food consumption in males across all dose levels between Days 1 and 4 of the pre-pairing phase compared to the control males. Males offered 1000 mg Isobutavan/kg body weight/day continued to consume less diet than the control males through the rest of the pre-pairing phase and throughout the post-pairing phase. There was no similar pattern for females during this phase.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
An assessment of the reproductive function revealed no adverse effects. Qualitative assessment of the testis did not indicate any abnormalities in the integrity of the various cell types present within the different stages of the spermatogenic cycle.

An assessment of the development of the first generation to Day 4 post-partum revealed no adverse effects.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
mortality

Target system / organ toxicity

Key result
Critical effects observed:
no
Lowest effective dose / conc.:
986.4 mg/kg bw/day (actual dose received)
Treatment related:
no
Dose response relationship:
no
Relevant for humans:
no

Any other information on results incl. tables

Stability: Diet formulations prepared at 300 and 25000 ppm showed stability over 24 hours when stored at room temperature, and over ten days when stored at

Homogeneity: The formulations prepared at 300 and 25000 ppm were deemed homogeneous. The samples from each concentration were within 10% of the mean and the coefficient of variation was ≤6.5% for both concentrations. Samples taken from formulations prepared for use on the first and last days of dosing also met the above criteria for homogeneity.

Concentration Verification: Diet formulations were considered acceptable for use. The mean results were within the range of 101 to 109% of nominal (target range 85 to 110% of nominal). Test article consumption was within 20% of the target dose with only a few exceptions, all of which involved consumptions greater than the target dose.

Applicant's summary and conclusion

Conclusions:
In conclusion, Isobutavan administered in the diet to male and female rats at 100, 300 or 1000 mg Isobutavan/kg bodyweight/day elicited no adverse effects.

Under the conditions of the current study, the no-observed-adverse-effect-level (NOAEL) for adult toxicity and reproductive and developmental toxicity was established as nominal 1000 mg Isobutavan/kg body weight/day (achieved mean high dose level of 986.4 mg/kg/day for males and 1215.3 mg/kg/day for females).
Executive summary:

The study was performed in accordance to the OECD 422 guidelines (Adopted 22 March 1996), which were current at the time this study was conducted. The dietary route of administration was chosen because it is an acceptable and commonly used route of exposure for regulatory studies of this type.

The test article was administered, ad libitum, in the diet to males for two weeks prior to pairing, during the pairing period and a further two weeks before necropsy. The males were provided with diet containing test article for a minimum of six weeks prior to necropsy. The test article was administered, ad libitum, in the diet to females for two weeks prior to pairing, during pairing and until Day 4 post-partum, inclusive. The females were allowed to litter and rear their offspring to Day 4 post-partum. The following were assessed: clinical observation, body weight, food intake, mating, litter data, functional observations and tests, locomotor activity, haematology, clinical chemistry, organ weights, gross pathology and microscopic pathology.

Diet formulations were considered acceptable for use. The mean results were within the range of 101 to 109% of nominal (target range 85 to 110% of nominal). Test article consumption was within 20% of the target dose with only a few exceptions, all of which involved consumptions greater than the target dose. All animals survived until their scheduled kill. There were no clinical observations that were considered to be related to the test article.

All animals gained weight over the duration of the study. Although there were some fluctuations and differences between groups in body weight gain, there was no clear dose relationship and these were considered most likely due to palatability of the diet containing Isobutavan and not indicative of toxicity. There was a dose-dependent decrease in food consumption in males across all dose levels between Days 1 and 4 of the pre-pairing phase compared to the control males. Males offered 1000 mg Isobutavan/kg body weight/day continued to consume less diet than the control males through the rest of the pre-pairing phase and throughout the post-pairing phase. There was no similar pattern for females during this phase. There was no effect on clinical pathology and no effect in the neuro-behavioural tests conducted. An assessment of the reproductive function revealed no adverse effects. Qualitative assessment of the testis did not indicate any abnormalities in the integrity of the various cell types present within the different stages of the spermatogenic cycle. There was no effect of Isobutavan on organ weights. Macroscopically, tissues were unremarkable and there were no microscopic findings indicative of Isobutavan-related effects. An assessment of the development of the first generation to Day 4 post-partum revealed no adverse effects.

In conclusion, Isobutavan administered in the diet to male and female rats at 100, 300 or 1000 mg Isobutavan/kg bodyweight/day elicited no adverse effects. Under the conditions of the current study, the no-observed-adverse-effect-level (NOAEL) for adult toxicity and reproductive and developmental toxicity was established as nominal 1000 mg Isobutavan/kg body weight/day (achieved mean high dose level of 986.4 mg/kg/day for males and 1215.3 mg/kg/day for females).

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