Dimethyl (p-methoxybenzylidene)malonate

Endpoint summary

Description of key information

A NOAEL of 300 mg/kg bw/day could be derived from a reliable combined repeated dose and reproduction screening study in rats.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Wistar rats - HsdHan: WIST rats; Conventionally bred (In-house random bred)

Sex:

male/female

Details on test animals or test system and environmental conditions:

No. of groups: 6
Main groups:
Vehicle control (G1)
Low dose (G2)
Mid dose (G3)
High dose (G4)
Recovery Groups:
Vehicle control recovery (G1R)
High dose recovery (G4R)
No. of animals for acclimatization: 60 males and 60 females
No. of rats/group: Main groups: 10 males + 10 females per group; Recovery groups: 5 males + 5 f
emales per group; Total = 100 (50 males + 50 females)
Age at treatment: 16-17 weeks
Body weight range at the start of treatment: Males: 370.61 to 478.55 g; Females: 235.23 to 310.02 g

Route of administration:

oral: gavage

Vehicle:

other: 1 % (w/v) Na CMC (medium viscosity) in Milli-Q water

Details on oral exposure:

Males: The dose formulation was administered to the rats of the specific groups once daily at appr
oximately the same time each day (varying by +/- 3 hours) for 2 weeks prior to mating and the treat
ment was continued during the mating period and approximately two weeks post mating.
Females: The dose formulations were administered to the specific group of rats once daily at ap
proximately the same time each day (varying by +/- 3 hours) throughout the treatment period. Treatm
ent was done 2 weeks prior to the mating period and continued through mating, pregnancy and up to
lactation day 13, after which, pups were sacrificed on lactation day 13 and parental females (dams)
were sacrificed on lactation day 14 after overnight fasting (water allowed).
The dose formulations were administered to the high dose recovery group of rats once daily at
approximately the same time each day (varying by +/- 3 hours).
The volume of dose administered was 10 mL/kg Bwt throughout the study. The dose volume was
adjusted based on the most recent body weight of individual rat.
Similarly, vehicle was administered to rats in the vehicle control and vehicle control recovery groups at
10 mL/kg Bwt.
The vehicle and the test item was not administered for vehicle control recovery and high dose
recovery groups, respectively for 14 days following the treatment period.
Homogeneity of the dose formulations during sampling/gavaging was maintained by constant stirring
using a magnetic stirrer.

Details on mating procedure are provided in chapter 7.8.1

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

DOSE FORMULATION PREPARATION:
The dose formulations were prepared daily prior to start of the treatment and prepared on need basis
and used within the stability period.
Following procedure was followed when 150 mL of dose formulation prepared:
The quantities of 1500 (G2), 4500 (G3) and 15000 (G4) mg of test item was weighed in a glass
beaker and transferred to the mortar. The test item was ground to a fine powder in the mortar using
pestle. Small volume of vehicle (1 % (w/v) NaCMC in Milli-Q water) was added in a stepwise manner
to mortar with continuous trituration till a uniform suspension was obtained. The micture was tr
ansferred into the measuring cylinder. Further, a snall volume of vehicle was added to the mortar and
rinse with vehicle to ensure the test item was completely transferred. The final volume was made up
with vehicle and to attain a desired concentration.
DOSE FORMULATION ANALYSIS:
For homogeneity and active ingredient (a.i.) concentration analysis, prepared formulation samples
were sampled in duplicate sets on Day 1 and once during Week 4 of the treatment and were sent to
Analytical R&D of Advinus Therapuetics Limited. For each set, duplicate samples were drawn from
top, middle and bottom layers of each preparation and in case of control, duplicate samples from
only middle layer was drawn.
The analysis was done as per the method validated under Advinus Study No. G9993. One set of s
amples were analyzed for concentration analysis and other set (second set) of samples were stored
at ambient conditions for reanalysis purpose as a backup and the second set of samples were
discarded, as the analysis results of first set of samples were within the limits.

Duration of treatment / exposure:

see 'Details on exposure'

Frequency of treatment:

see 'Details on exposure'

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Vehicle control group (G1)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Low dose group (G2)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Mid dose group (G3)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

High dose group (G4)

No. of animals per sex per dose:

10 males + 10 females per group

Control animals:

yes, concurrent vehicle

Details on study design:

SELECTION OF DOSE LEVELS AND DOSE JUSTIFICATION
Three dose levels of 100 (low dose), 300 (mid dose) and 1000 (high dose) mg/kg bw/day were selected for groups G2, G3 and G4 respectively as recommended by the sponsor.
In addition to the test doses, vehicle control and vehicle control recovery groups were included. Animals in the vehicle control and recovery were handled in a manner similar to the treatment groups
except for test item administration

Observations and examinations performed and frequency:

Clinical Signs, Morbidity and Mortality
All rats were observed for morbidity and mortalities twice daily i.e., once in the morning and once in the afternoon. As there were no clinical signs, the observation for morbidity and mortality was carried
out once in the morning during holidays. All rats were observed for clinical signs once daily during the treatment and recovery periods.

Detailed Clinical Examination
Detailed clinical examination was done prior to the treatment on Day 1 and at weekly intervals thereafter during treatment and recovery periods. During detailed clinical examination, all rats were observed for changes in skin, fur, eyes, mucous
membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling or bizarre behaviour like self-mutilation, walking backwards. On the days of detailed clinical examination, general clinical signs were not performed except on
treatment Day 1.

Functional Observation Battery Tests (FOB)
The following neurological examination was conducted for randomly selected 5 males and 5 females in each group at termination i.e. towards the end of the dosing period for males (shortly prior to their scheduled kill) and during lactation period for females (shortly before the scheduled kill). For recovery groups, neurological examination was carried out towards the end of recovery period.

Home Cage Observations
Each rat was observed in the home cage for posture and for presence or absence of abnormal vocalizations and convulsions.

Observations during Removal of Animal from Home Cage and Handling
The objective of this phase of neurological examination was to observe the subject's response to handling and to conduct other procedures of the FOB that can best be performed when the rat is being
held. Each rat was observed for the following examinations:
- ease of removal from home cage
- handling reactivity
- palpebral closure
- eye examination
- piloerection
- lacrimation
- salivation
- skin/fur examination
- perineum wetness
- respiration
- muscle tone and
- extensor thrust response
The observations were recorded using scores/ranks.
Open Field Observation
Rat was placed (one at a time) in an open arena, on a flat surface with a clean absorbent paper and
observed for at least 2 minutes. Absorbent paper was replaced for each rat. During this observation
period, rat was evaluated as it moves about freely/unperturbed and the following observations were
made and observations were recorded using score/ranks:
- gait
- posture
- tremors
- mobility score
- arousal level
- clonic or tonic movements
- stereotypic behaviour
- urination
- defecation
- rearing
- abnormal vocalizations

Functional Tests
Functional testing included motor activity, sensory evaluation, landing hindlimbs footsplay and measurement of grip performance.

Motor Activity
The motor activity of rats was measured using an automated animal activity measuring system (Make: Columbus Instruments) equipped with a computer analyzer. Each rat was individually placed in the activity cages of the instrument. The rats were monitored for 30 minutes. During this motor activity measurement session, parameters viz., the stereotypic time (small movements) in secobds, the ambulatory time (large ambulatory movement) in seconds, horizontal counts, ambulatory counts
were monitored. The Opto-Varimex 4 motor activity measurement system provides the data at 1 minute interval and the data was analyzed in blocks of 10 minutes interval and the same was reported.

Sensory Reactivity Measurements
After the 2 minutes (approximately) observation period, while the rat is in the open field arena, the following tests were conducted. The rat was allowed to move freely in the open field box for these tests but positioned in the box by the observer in order to administer stimulus. During sensory reactivity measurements, rats were observed for following and the observations were recorded using scores/
ranks.
- approach response
- touch response
- click response
- tail-pitch response
- pupil response
- aerial righting reflex
Landing Hindlimbs Footsplay
The landing hind limbs foot splay was performed by dropping the rat on to a horizontal surface of the table top from a short height and measuring the distance between the hind feet upon landing. The heel portion of each hind foot of each rat was marked with non-permanent ink or the hind feed of the rat was gently pressed to an ink pad just prior to testing. The rat was suspended in a prone position and then dropped from a height of approximately 30 cm on to a SOP format, which has the details such as
Study No., Animal No., Group and sex. A clean recording paper sheet was used for each rat. A total of 3 readings were recorded for each rat and average of 3 footsplay values were presented in the report.

Grip performance
Hindlimbs and forelimbs grip performance was tested using computerized dual grip strength meter (Model: Columbus Instruments). At least three trials were conducted for each rat i.e., three trials for forelimb and hind limbs. Average of three trials for both forelimb and hindlimbs were calculated and presented in the report.

Physiological Observations
Body temperature (rectal temperature) was measured in Celcius (°C) using digital thermometer. At the end of the functional test, body weight of each rat was measured.

Body weights
i) Individual body weights of males were recorded initially and at weekly intervals thereafter. Individual body weights of females were recorded initially and at weekly intervals thereafter till cohabitation (till mating confirmation) with males.
ii) All dams were weighed on Gestation Days (GD) 0, 7, 14 and 20 and on Lactation Days (LD) 0, 4 and 13.

Food consumption
Cagewise food consumption was calculated by using the food consumed at weekly interval per cage and dividing by the number of rats per cage and the number of days in the intervening period to determine the food intake/rat/day.
Food consumption was not measured during the cohabitation period. Food consumption of pregnant dams was recorded on GD 7, 14 and 20 and on Day 4 and 13 of lactation period.

Parturition
The duration of gestation was calculated from Day '0' of pregnancy to the day of parturition (Gestation Length). Females were observed for signs of difficult or prolonged parturition.

Hormone Analysis
Blood samples were collected and serum was separated as per the following schedule for the determination of total T4 (Thyroxin) and thyroid stimulating hormone (TSH):
- Two pups per litter on Day 4 after birth
- All dams at termination on Day 14
- Two pups per litter on Day 13 and
- All adult males, at termination.
The blood from pups was pooled by litter for thyroid hormone analysis. Pups were lightly anesthetized with isoflurane and incised at jugular vein in the neck region. The collected samples were pooled together for each litter. Blood samples were collected in plain labeled tubes and kept on bench top for 15 -20 min before centrifugation. Serum was separated by centrifuging the whole blood samples at 5000 rpm for 5 minutes at 4 °C. the serum samples were placed in labeled
plastic tubes and stored at ~-70 °C until they were analyzed.

Clinical Pathology (i.e. Blood Collection, Haematology, Coagulation Parameters, Clinical Chemistry,
Urinalysis, Thyroid Profile Hormones) as well as Anatomic Pathology (i.e. Necropsy, Tissue Collection,
Histopathology) were also assessed within this study.

Sacrifice and pathology:

Postmortem examinations (parental animals)
All adult animals were examined macroscopically for any structural abnormalities and pathological changes. The adult animals sacrificed at term were fasted overnight (water allowed), exsanguinated under isoflurane anaesthesia and weighed. The rats were subjected for detailed necropsy by a veterinary pathologist and findings were recorded. The number of implantation sites was recorded for all the dams.

Statistics:

The statistical analysis of the experimental data was carried out using the validated package in excel and also using licensed copies of SYSTAT Statistical package ver. 12.0. All quantitative variables like body weight, food intake, terminal body weight, clinical pathology (haematology clinical chemistry a nd coagulation) parameters, thyroid hormone levels, organ weights and organ weight ratios were tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene's test) within the group before performing a one-factor analysis of variance (ANOVA) modeling by treatment groups. Nonoptimal (non-normal or heteroscedastic) data were transformed, before using ANOVA. Means between treatment groups and vehicle control group were compared using Dunnett's test. Pre-implantation loss (%), post-implantation loss (%), no. of corpora lutea, implantations, pre-coital interval, sex ratio (%) and gestation length (days) were analysed after suitable transformation of the data. One-way ANOVA was carried out for the transformed data. Dunnett's pair-wise comparison of the treated means with the control mean was done for the significant group differences. Z test was performed for testing the differences in proportions for mating and fertility indices.

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs were observed at any of the doses tested. However, clinical sign of sparse hair loss at right and left flank was observed in a female (Rt1396) of control group and one male rat (Rt1473) of high dose recovery group was considered spontaneous finding and not related to treatment.

Mortality:

no mortality observed

Description (incidence):

No mortality was observed at any of the doses tested.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males, body weights: No treatment related variations in the mean body weights at all the dose groups during the treatment period. The mean body weight was significantly lower in high dose recovery animals.
Females, body weights: No treatment related variations in the mean body weights at all the dose groups either during the treatment period or recovery periods.
Maternal Body Weights and Food Intake during the Gestation Period: No significant changes in maternal body weight and food consumption were observed at any of the doses tested when compared to the vehicle control group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males: The Food consumption was unaffected by the treatment at 100 mg/kg bw/day dose groups. At 300 mg/kg bw/day, the food consumption was significantly lower on week 6 and was considered in cidential because of isolated occurrence and there was no associated change in the body weight. At 1000 mg/kg bw/day, the food consumption was significantly lower and considered treatment related finding. In the 1000 mg/kg bw/day dose recovery group, the food consumption was also significantly lower on weeks 2 and 3 and hence were considered to be transient and not of biological significance.
Females: The food consumption was unaffected by the treatment at 100, 300 and 1000 mg/kg bw/day dose groups. In the high dose recovery group, the food consumption was significantly lower on weeks 1 and 3 and was considered incidential because of isolated occurrence and there was no associated change in the body weight.

Haematological findings:

no effects observed

Description (incidence and severity):

The test item administration did not reveal any treatment related changes in the hematology parameters of both male and female rats.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

The test item administration did not reveal any treatment related changes in the clinical chemistry parameters of both male and female rats.

Urinalysis findings:

no effects observed

Description (incidence and severity):

The test item administration did not reveal any treatment related changes in the urinalysis parameters of both male and female rats.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Males and females: Home cage, Handling, Open field and Sensory observations: No treatment-related abnormalities were observed in all the tested dose groups either during treatment period or recovery periods.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

At 1000 mg/kg bw/day, the test item-related changes included the following: Decreased terminal fasting body weight in males, decreased prostate and seminal vesicle with coagulating gland weights without any associated microscopic changes, considered secondary to reduction in body weight; increased kidney weight in males.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, the test item-related changes included the following: grossly white foci in nonglandular mucosa of stomach in male rats. There were no test item-related changes in male and female reproductive organs.

Neuropathological findings:

no effects observed

Description (incidence and severity):

MALES:
-Neuromuscular observations: No treatment-related variations in the hind limb footsplay and forelimbs and hind limbs grip strength either during the treatment period or recovery periods.
-Motor activity: The following statistically significant variations were observed in the motor activity of rats when compared to respective vehicle control group:
Lower: Stereotypic time at interval 1, interval 3 and total at the mid dose; Ambulatory time at interval 1 at the high dose recovery. The above observed statistical variations in the motor activity measurement were considered to be incidental as the observed changes did not follow the dose proportion and there were no changes in the home cage of open field observations.
FEMALES:
- Neuromuscular observations: No treatment-related variations in the hind limb footsplay and forelimbs and hind limbs grip strength either during the treatment period or recovery periods. However, higher hindlimbs footsplay value was observed at mid dose group and was considered incidential be cause of the lack of any dose relationship.
- Motor activity: No treatment-related changes were observed in the motor activity at all the tested
doses.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related histopathological changes were observed in stomach (hyperkeratosis and epithelial hyperplasia in nonglandular mucosa; and mucosal necrosis) and kidneys (dilated tubules and tubular degeneration/regeneration) in both sexes at 1000 mg/kg bw/day, This change showed complete recovery at the end of recovery period. There were no test item-related changes in male and female reproductive organs.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

The mean oestrous cycle length in the treated groups was not significantly different from the vehicle control group.
No treatment-related effects were observed with respect to mean pre-coital time, gestation length and fertility indices when compared to control means.
A significantly lower female fertility index at 100 and 1000 mg/kg bw/day doses was considered toxicologically insignificant due to lack of dose relationship.
No treatment-related effects were observed with respect to corpora lutea, implantations, and percentage of pre and post implantation losses when compared to control means.

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Dose descriptor:

LOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (actual dose received)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (actual dose received)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

Under the conditions of this OECD 422 study, the no observed adverse effect level (NOAEL) for systemic toxicity is considered to be 300 mg/kg bw/day.

Executive summary:

In this GLP-compliant study according to OECD guideline 422 the test item was administered daily (gavage) to male and female Wistar rats at dose levels of 100, 300 and 1000 mg/kg bw/day for 2 weeks prior to mating, during mating, and 2 weeks post mating (males) or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery (females).

Based on the changes observed in both sexes at 1000 mg/kg bw/day, i.e. reduced body weights and food consumption, prostate and seminal vesicle with coagulating gland weights, increased kidney weight and grossly white foci in nonglandular mucosa of stomach in male rats, microscopic changes in stomach (hyperkeratosis and epithelial hyperplasia in nonglandular mucosa and mucosal necrosis) and kidneys (dilated tubules and tubular degeneration/regeneration)  the no observed adverse effect level (NOAEL) for systemic toxicity is considered to be 300 mg/kg bw/day.

The test item did not induce any adverse effects on fertility and reproductive perfomance.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Reliable study, GLP-compliant and according to OECD guideline 422

System:

other: gastrointestinal and urinary tract

Organ:

kidney

stomach

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

Mode of Action Analysis / Human Relevance Framework

In the absence of any evidence for species specific effects or modes of action the observed effects are regarded as relevant for humans.

Additional information

In this GLP-compliant study according to OECD guideline 422 the test item was administered daily (gavage) to male and female Wistar rats at dose levels of 100, 300 and 1000 mg/kg bw/day for 2 weeks

prior to mating, during mating, and 2 weeks post mating (males) or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery (females).

Based on the changes observed in both sexes at 1000 mg/kg bw/day, i.e. reduced body weights and food consumption, prostate and seminal vesicle with coagulating gland weights, increased kidney weight

and grossly white foci in nonglandular mucosa of stomach in male rats, microscopic changes in stomach (hyperkeratosis and epithelial hyperplasia in nonglandular mucosa and mucosal necrosis) and kidneys

(dilated tubules and tubular degeneration/regeneration) the no observed adverse effect level (NOAEL) for systemic toxicity is considered to be 300 mg/kg bw/day.

Justification for classification or non-classification

As adverse effects have been observed only at the highest dose (1000 mg/kg bw/day), no classification is required with respect to specific organ toxicity after repeated exposure according to the criteria laid down in Regulation (EC) No 1272/2008 (CLP).