3-{3-[(3,5,5-trimethylhexanoyl)oxy]-2,2-bis({[(3,5,5-trimethylhexanoyl)oxy]methyl})propoxy}-2,2-bis({[(3,5,5-trimethylhexanoyl)oxy]methyl})propyl 3,5,5-trimethylhexanoate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 February 2015 to 04 December 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

for 1 female in group 1, bodyweight and food consumption were recorded on Day 1 p.c.Proof of mating on the first day of estrous evaluation (Day 0 p.c.). Not thought to impact the integrity of the results

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

613-848-7

EC Number:

613-848-7

Cas Number:

65870-94-2

Molecular formula:

C33H62O6

IUPAC Name:

613-848-7

Details on test material:

- Physical state: Clear viscous fluid
- Storage condition of test material: Room temperature protected from light

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- A total of 124 Sprague-Dawley RjHan:SD rats (62 males and 62 females) were received on 03 March 2015.
- On the first day of treatment, males were approximately 10 weeks old and females were approximately 9 weeks old.
- At the start of treatment, mean body weight was 422 g (range: 375 g to 458 g) for males and 255 g (range: 224 g to 277 g).
- Males and females were sexually mature and were not siblings.
- Females were virgin.
- Animals were clinically examined on arrival to ensure they were in good health.
- The animals were acclimated to the study conditions for a period of 8 days before the beginning of the treatment period. A larger number of animals than necessary were acclimated to permit selection and/or replacement of individuals.
- During the acclimation period, the required number of animals (60 males and 60 females) was selected according to body weight and clinical condition. The animals were allocated to groups (by sex) using a computerised randomisation procedure based on body weight so that the average body weight of each group was similar.
- Each animal was identified by an individual ear tattoo or on the tail. At the beginning of the study, each animal received a unique identity number.

ENVIRONMENTAL CONDITIONS
- Animals were housed in a barriered rodent unit from the point of receipt.
- Temperature: 22 ± 2 °C
- Relative humidity: 50 ± 20 %
- Lighting: 12 hours light and 12 hours dark
- Ventilation: 8 to 15 changes/hour of filtered, non-recylced, air
- The corresponding instrumentation and equipemtn were checked and calibrated at regular intervals.
- Temperature and relative humidity were recorded continuously (recording devices equipped with alarm systems).
- The animal room was disinfected before the arrival of the animals and cleaned regularly thereafter.

HOUSING
- Except during pairing and lactation, animals were individually housed in polycarbonate cages (Tecniplast 2154, 940 cm2) with stainless steel lids and containing autoclaved sawdust (SICSA, Alfortville, France). Individual housing was chosen in order to not jeopardise gestations and to avoid aggressive behaviour around mating between males
- Towards the end of gestation and during lactation, autoclaved wood shavings (SICSA, Alfortville, France) were placed in each cage of females.
- Each cage contained an object (rat hut) to provide environmental enrichment.
- The cages were placed in numerical order on the racks.

FOOD AND WATER
- All animals had free access to SSNIFF R/M-H pelleted maintenance diet (batch 4482794; SSNIFF Spezialdiaten GmbH, soest, Germany), which was distributed weekly.
- Animals had free access to bottles containing tap water (filtered with a 0.22 µm filter).

CONTAMINANT ANALYSES
- Batches of diet, sawdust and wood shaving were analysed by the suppliers for composition and contaminant levels.
- Bacterial and chemical analyses of water were performed regularly by external laboratories. These analyses included the detection of possible contaminants (pesticides and heavy metals).
- No contaminants were present in the diet, drinking water, sawdust or wood shavings at levels which could be expected to interfere with or prejudice the the outcome of the study.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

(Batch number MKBQ9948V)

Details on exposure:

DURATION
- Dose formulations were administered to males daily for 2 weeks before mating, during the mating period and until sacrifice or treatment-free period (at least 5 weeks of treatment in total).
- Dose formulations were administered to females for 2 weeks before mating, during the mating period, during gestation, during lactation until day 5 post-partum inclusive or until sacrifice for females with no delivery.
- At the end of the treatment period, the animals were sacrificed expect for certain animals in groups 1 and 5 (the last five surviving males and the first five surviving lactating females). Animals that were not sacrificed were kept for a 2 week treatment-free period.

ADMINISTRATION
- Dose formulations were administered once daily, at approximately the same time of day, by gavage using a plastic syringe fitted with a metal gavage tube.
- The quantity of dose formulationadministered to each animal was adjusted according to the most recently recorded body weight.
- A constant dose volume of 4 mL/kg bw/day was used.
- Control animals (group 1) received the vehicle alone.
- The control dose formulation was stirred just before administration and the test item dose formulations for 15 minutes before administration.
- Formulations were maintained under continuous magnetic stirring throughout the dosing procedure.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Gas Chromatography with FID detection (GC-FID)
- Analytical method provided by the sponsor and validated prior to dose formulation analysis.
- Test item concentration in dose formulations was determined once on formulations used in weeks 1, 3 and 6. A sample was taken from control and test item dose formulations and analysed using the validated method.
- Measured test item concentration was found to equal nominal concentration ± 10 %.

Details on mating procedure:

MATING
- Females were paired with males from the same dose-level group.
- One female was placed with one male in the latter's cage during the night.
- Confirmation of mating was made in the morning by checking for the presence of a vaginal plug or for sperm in a vaginal lavage.
- The day of confirmed mating was designated day 0 post-coitum.
- Each female was placed with the same male until mating occurred.
- The pre-coital time was calculated for each female.

PARTRUITION
- Females were allowed to litter normally and rear their progeny until day 5 post-partum.
- Any sign of a difficult or prolonged partruition was recorded.
- The morning when partruition was completed was designated day 1 post-partum.
- The length of gestation was calculated.

Duration of treatment / exposure:

- Males: 2 weeks before mating, during the mating period, then until sacrifice or treatment-free period (at least 5 weeks)
- Females: 2 weeks before mating, during gestation, during lactation until day 5 post partum (inclusive). Females that didn't deliver were dosed until sacrifice.

Frequency of treatment:

Once daily

Duration of test:

61 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

250 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

- Group 1: 0 mg/kg bw/day (15 males including 5 treatment free males and 15 females including 5 treatment-free females )
- Group 2: 100 mg/kg bw/day (10 males and 10 females)
- Group 3: 250 mg/kg bw/day (10 males and 10 females)
- Group 4: 500 mg/kg bw/day (10 males and 10 females)
- Group 5: 1000 mg/kg bw/day (15 males including 5 treatment free males and 15 females including 5 treatment-free females )

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Details on study design:

ESTROUS CYCLICITY (Parental animals)
- The estrous cyclce stage was determined from a fresh vaginal lavage (stained with methylene blue).
- The procedure was repeated each morning during the mating period until the females were mated.

SPERM PARAMETERS (Parental animals)
- During microscopic examination following sacrifice, special emphasis was placed on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Examinations

Maternal examinations:

CLINICAL EXAMINATIONS (F0 ANIMALS)

Morbidity and mortality: Each animal was checked for mortality or signs of morbidity once a day before the treatment period and at least twice a day during the treatment and treatment-free periods (including weekends and public holidays).

Clinical signs: From arrival, each animal was observed once a day as part of routine examinations. From the start of the treatment period, each animal
was observed once a day, at approximately the same time each day, for the recording of clinical signs.

MEDICAL CARE
- Cothivet, an antiseptic and healing product, was applied on lesions of animals D27526 (group 1), D27474 (group 3) and D27477 (group 4) for 2 or 5 days.

BODY WEIGHT

- The body weight of each male was recorded on the first day of treatment (day 1) and then once per week until sacrifice.
- The body weight of each female was recorded on the first day of treatment (day 1), then once a week until mated, on days 0, 7, 14 and 20 post-coitum, and on days 1 and 5 post-partum. Body weights of treatment-free females were recorded on days 12 and 19.

FOOD CONSUMPTION
- The quantity of food consumed by each male was measured once a week over a 7 day period from the first day of treatment until the start of the mating period, and then once a week during the treatment-free period for treatment-free males.
- The quantity of food consumed by each female was measured once a week, over a 7 day period, from the first day of treatment until the start of the mating period, during gestation for the intervals days 0-7, 7-14 and 14-20 post-coitum, and during lactation days 1-5 post-partum. Food consumption of treatment-free females was also measured during days 5-12 and 12-19 post-partum.
- Food consumption was not measured for males or females during the mating period.
- Food intake per animal and per day was calculated by noting the difference between the food given and that in the food hopper the next time.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day: Day 5 post partum
- Organs examined: All animals were observed for macroscopic lesions
The kidneys, livers and spleens were weighed for all animals.
The mammary gland area, ovaries (with oviducts), uterus (horns and cervix), vagina, kidneys, liver and spleen were preserved for all animals.
Microscopic examinations of the ovaries, kidneys and livers were performed for all control and high dose animals.

Ovaries and uterine content:

Special attention was paid to the reproductive organs. The numbers of corpora lutea and implantation sites were also recorded for females sacrificed as scheduled on Day 6 p.p. and on Day 20 p.p. (except the number of corpora lutea on this latter day) and for females sacrificed on Days 25-26 p.c. due to no delivery.
For non-pregnant females the absence of implantation scars on the uterus was checked using the ammonium sulphide staining technique (Salewski, 1964).

Statistics:

Data were expressed as group mean values ± standard deviation (body weight, body weight change, food consumption, number of corpora lutea, number of implantation sites, number of pups and pup body weight , gestation length) or as ratios (pre-implantation loss, post-implantation loss, pup observations, mating index, fertility index, gestation index, live birth index, viability index).
Data for non-pregnant females are not included in group mean calculations such as body weight, body weight change, food consumption

Body weight, food consummation and reproductive data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fischer exact probability test (ratios).

Organ weight data was assed using PathData software (level of significance 0.05 or 0.01) according to the following:
Kolmogorov test for normality was performed on each group. Where evidence for non-normal distribution occurred in one group or more, non-parametric test for groups differences (Kruskal-Wallis) was performed if the number of groups was greater than 2. The data were then reviewed using Dunn’s test when evaluating more than 2 groups, or Wilcoxon test, where 2 groups were examined.
If the Kolmogorov test for normality did not demonstrate non-normal distribution occurred in one group or more, the data were analysed of homogeneity of variance (Bartlett test/ F-test). Where a high homogeneity of variance was observed, the data were evaluated using a parametric test for group differences in means using one-way analysis of variance if more than 2 groups. Further analysis was performed using Dunnett’s test for more than 2 groups, or t-test if 2 groups.

Indices:

Pre-implantation loss: ((Number of corpora lutea – Number of implantation sites)/Number of corpora lutea) x 100

Post implantation loss (Calculated manually): ((Number of implantation sites – Number of live pups)/Number of implantation sites) x 100

Mating index: (Number of mated animals/Number of paired animals) x 100

Fertility index: (number of pregnant female partners/number of mated pairs) x 100

Gestation index: (Number of females with live born pups/Number of pregnant females) x 100

Live birth index: (Number of live born pups/Number of delivered pups) x 100

Viability index and Day 4 p.p.: (Number of surviving pups on Day 4 p.p./Number of live born pups) x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

- The only test item-related clinical sign was ptyalism noted in 4/15 males at 1000 mg/kg bw/day. In view of the low incidence (4/30 animals in the high dose group) and the absence of a similar finding in females, it was considered to be of no toxicological significance.

- Other clinical signs recorded in test item-treated groups (ptyalism, chromodacryorrhea, hair loss, cutaneous lesion, reddish vaginal discharge, piloerection, pallor) were not attributed to the test item treatment as there was no dose relationship and observations were recorded in only a few animals or in a similar incidence to that in controls.

Mortality:

no mortality observed

Description (incidence):

There were no premature deaths in test item-treated groups.

Control female D27529 was prematurely sacrificed on day 22 post-coitum due to the presence of a mass in the urogenital region that made a normal delivery impossible. Swollen urogenital area was noted from day 12 post-coitum and piloerection, round back, hypoactivity and absence of feces were recorded on day 22 post-coitum. Another mas, in the thoracic region, was noted in this female from day 9 post-coitum. The two large masses correlated with mammary gland adenocarcinoma. This finding is not uncommon in female rats of this strain and age.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

BODY WEIGHT - Mean body weights and mean body weight changes (g) are summarised in the tables attached. - There were no effects on mean body weights and mean body weight changes in males at any dose levels during treatment and treatment-free periods. The statistically significant higher mean body weights gain at 250 mg/kg bw/day over the first 15 days of treatment in males was considered to be incidental and not dose-related. - There were no toxicologically significant effects on mean body weight in females at any dose levels during treatment and treatment-free periods. - Statistically significant lower mean body weight gains were recorded at 500 and 1000 mg/kg bw/day in the second week of treatment, and statistically significant higher mean body weight gains were recorded at 1000 mg/kg bw/day in the first week of the treatment-free period. As the effects were transient and did not have significant impact on mean body weights, they were considered to be of no toxicologicical significance.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no toxicologically significant effects on mean food consumption during treatment and treatment-free periods.

At 1000 mg/kg bw/day, the statistically higher mean food consumption in females during the first week of treatment and the last week of gestation was considered to be of no toxicological significance. The increases were small, transient and more variable during the first week of treatment than in controls. Moreover, there was no relevant impact on mean body weights.

At 500 mg/kg bw/day in males, the statistically significant lower mean food consumption during the second week of treatment was not ascribed to treatment with the test item as it was not dose related.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At the end of the treatment period there were test item-related liver and kidney mean weight differences in males

The final mean body weights were considered to be unchanged by test item administration.

Moderately increased, slightly dose-related, absolute and relative-to-body mean kidney weights were noted in males at all dose levels (up to +34 % in absolute weights with P < 0.01 in males at 1000 mg/kg bw/day. These changes were considered to be related to test item administration since they correlated with the hepatocellular hypertrophy and the increased periportal vacuolation seen microscopically.

It was considered that the minimally higher liver relative-to-body weights in females at 250 and 500 mg/kg bw/day were probably unrelated to test item administration since those changes were poorly dose related and of very low magnitude. The slides of liver from females were not examined. The minimal epididymides and spleen absolute and relative-to-body mean weight changes in males at all dose-levels were not considered to be test item-related because they were of small magnitude, not dose-related and uncorrelated with microscopic findings. At the end of the treatment-free period there were no mean liver weight differences while minimally increased kidney weights were seen in males previously treated at 1000 mg/kg bw/day (+17 % in relative-to-body weights with p < 0.05), with microscopic correlates as mentioned at the end of the treatment period. This suggested total reversibility for the liver changes but not for the kidney changes for which reversibility was incomplete.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

There was one unscheduled death when one control female was sacrificed with two large subcutaneous white masses (3 x 4 and 2 x 3 cm) which correlated with mammary gland adenocarcinoma.

At the end of the treatment period there were test item-related irregulat colour and/or tan discolouration in the kidneys from 6/10 males treated with 100 mg/kg bw/day and from all males treated at 250, 500 or 1000 mg/kg bw/day. These changes were considered to be related to test item administration since they correlated with the increased weights and hyaline droplets in tubular epithelium, tubular dilation and dilated tubules with eosinophilic content seen microscopically in the kidneys of these males.

There were increased incidence of accentuated lobular pattern in the liver from males treated at 100, 250, 500 or 1000 mg/kg bw/day, together with tan discolouration. These findings correlated with the increased weights and the hepatocellular hypertrophy plus the increased periportal vacuolation seen microscopically in these animals.

The few other gross observations were considered to be consistent with spontaneous findings encountered in the rats of this strain and age because they were not dose-related, isolated and/or correlated with common background lesions.

At the end of the treatment-free period there were no test item-related macroscopic findings, suggesting a complete reversibility of these changes

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

. Microscopic examination The control female sacrificed on day 38 had a mammary gland adenocarcinoma. This finding is not uncommon in female rats of this strain and age. At the end of the treatment period a series of dose-related test item-related microscopic findings were seen in the kidneys and liver of males treated from 100 mg/kg bw/day, which were considered adverse at 1000 mg/kg bw/day.

Kidney - Minimal degeneration/necrosis in the kidneys from two males treated at 1000 mg/kg bw/day, suggesting an adverse effect at this dose level. - Increased incidence and severity of minimal to moderate tubular basophilia at all dose levels, considered not to be adverse in view of the low magnitude of the increase. - Minimal to marked hyaline droplets in tubular epithelium in all test item-treated males, considered to be a species-specific change not relevant in humans. - Minimal to slight tubular dilation (i.e. with empty lumen), together with minimal to moderate dilated tubules containing a granular eosinophilic content, considered not to be adverse in view of the absence of associated degeneration/necrosis. - Increased incidence of minimal infiltrate of mononuclear cells in males treated at 1000 mg/kg bw/day although poorly dose-related, considered not to be adverse in view of the minimal severity of this change.

Liver - Minimal hepatocellular hypertrophy, considered not to be adverse in view of the minimal severity of this change. - Increased incidence and severity of minimal to slight periportal vacuolation, considered not to be adverse in view of the low magnitude of this increase. These findings correlated with gross irregular colour and tan discolouration in kidneys and accentuated lobular pattern with tan discolouration in liver, together with increased weights. There were no test item-related findings in the testes, epididymides or ovaries at microscopic examination. There were a few other microscopic observations, which were seen at low incidence, at minimal severity, were not dose-related and correlated with background lesions seen in rats of this strain and age. At the end of the treatment-free period there were no test item-related findings in the liver while lesions similar to those recorded at the end of the treatment period were seen in the kidneys and correlated with the increased mean renal weights. - Increased incidence and severity of slight to marked tubular basophilia. - Minimal hyaline droplets in tubular epithelium from one test item-treated male. - Increased severity of minimal to moderate tubular dilation (i.e. with empty lumen) together with slight to moderate dilated tubules containing a granular eosinophilic content. - Increased incidence of minimal infiltrate of mononuclear cells. These findings suggested complete reversibility for the liver changes while there was incomplete reversal of the renal findings.

Histopathological findings: neoplastic:

no effects observed

Maternal developmental toxicity

Pre- and post-implantation loss:

no effects observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

- Control females D27525, D27526 and D27533 and female D27544 from the low dose group were sacrificed on days 25/26 post-coitum because they did not deliver (not ascribed to test item treatment for D27544 and the other animals were controls). The animals were found to be non-pregnant except control female D27525 which had one late resorption in the uterus at necropsy. There were no test item-related effects on mean delivery data at any dose levels.

Changes in pregnancy duration:

not specified

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

There were no effects on mean pairing, mating and fertility data at any dose level.

Details on maternal toxic effects:

Maternal toxic effects:no effects
- The higher mean number of corpora lutea at 500 mg/kg bw/day was due to female D27557 with an excessive number of corpora lutea. As this was isolated, the finding was considered to be incidental. Nevertheless, the very high number of corpora lutea (40) likely did not correspond to gestational corpora lutea only (total number abnormally high).
-Female D27537 (100 mg/kg bw/day) and females D27557 and D27564 (500 mg/kg bw/day) were blocked in diestrous during most of the mating period and thus took more days to mate compared with the other animals. This was not considered to be related to test item treatment as it was not dose related and occurred in a limited number of animals.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Remarks:

females

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: other:

2

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

3

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no toxicologically significant effects on pup mean body weight and mean body weight gains at any dose-levels during treatment and treatment-free periods.

At 1000 mg/kg/day and when compared with controls, the slightly higher mean body weight gains and mean body weights during the treatment-free period was mainly due to the lower mean number of pups kept after the end of treatment of their mothers.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

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:

other: absence of adverse effects in pups at all dose levels

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

CHEMICAL ANALYSIS OF DOSE FORMULATIONS
- Test item concentrations in the administered dose formulations analysed in weeks 1, 3 and 6 were within an acceptable range of variation (-4.9 % to +3.9%) when compared to nominal values (± 10 % of nominal concentration).
- No test item was observed in the control dose formulation in week 1 although a small peak (below the limit of quantification) was observed in weeks 3 and 6.

Table 1: Parental Bodyweight and Mean Body Weight Changes (g)

Sex	Male					Female
Dose level (mg/kg bw/day)	0	100	250	500	1000	0	100	250	500	1000
Premating or whole study	428	430	416	409*	423	252	255	251	255	259
Day 1	462	470	458	450	463	263	266	259	265	273
Day 8	495	506	493	484	494	282	279	273	275	282
Day 15	506	520	509	497	510	/	/	/	/	/
Day 22	526	546	538	525	532	/	/	/	/	/
Day 29	535	546	535	522	534	/	/	/	/	/
Day 35	580	/	/	/	553	/	/	/	/	/
Day 43 (treatment free)	566	/	/	/	545	/	/	/	/	/
Day 50 (treatment free)	+34	+40	+42	+41	+40	+10	+11	+8	+10	+14
Days 1-8	+33	+36	+35	+34	+31	+19	+13	+14	+10*	+9**
Days 8-15	+67	+76	+77*	+75	+71	+29	+23	+22	+20	+23
Days 15-22	+11	+14	+16	+13	+16	/	/	/	/	/
Days 22-29	+20	+26	+29	+28	+23	/	/	/	/	/
Days 29-36	+9	0	-3	-3	+2	/	/	/	/	/
Day 15-36	+40	+40	+42	+38	+41	/	/	/	/	/
Days 1-36	+107	+116	+119	+114	+111	/	/	/	/	/
Day 36-43	+13	/	/	/	+13	/	/	/	/	/
Days 43-50	-13	/	/	/	-8	/	/	/	/	/
Days 36-50	-1	/	/	/	+4	/	/	/	/	/
Days 1-50	+134	/	/	/	+131	/	/	/	/	/

 

Table 2:Female bodyweight changes Day 0 p.c. to Day 19 p.p.

Dose level (mg/kg bw/day)	0	100	250	500	1000
Gestation
Day 0 p.c.	284	287	276	286	282
Day 7 p.c.	318	323	314	318	321
Day 14 p.c.	358	366	357	361	363
Day 20 p.c.	437	452	452	452	449
Day 0-7 p.c.	+34	+35	+35	+31	+38
Day 7-14 p.c.	+39	+43	+43	+43	+43
Day 14-20 p.c.	+80	+86	+86	+91	+86
Day 0-20 p.c.	+153	+164	+164	+165	+167
Lactation
Day 1 p.p	351	355	341	345	346
Day 5 p.p	358	365	356	351	349
Day 12 p.p (treatment free)	377	/	/	/	380
Day 19 p.p (treatment free)	383	/	/	/	371
Days 1-5 p.p	+7	+10	+15	+6	+3
Days 5-12 p.p	+29	/	/	/	+49*
Days 12-19 p.p	+5	/	/	/	-9
Days 5-19 p.p	+34	/	/	/	+41
Days 1-19 p.p.	+35	/	/	/	+39
* p < 0.05

 

Table 3: Meanparental food consumption (g)

Sex	Male					Female
Dose level (mg/kg bw/day)	0	100	250	500	1000	0	100	250	500	1000
Pre-mating or whole study
Days 1-8	31	32	32	31	33	19	20	19	20	23*
Days 8-15	31	30	30	29**	30	21	20	19	19	20
Days 36-43 (treatment free)	33	/	/	/	31	/	/	/	/	/
Days 43-50 (treatment free)	29	/	/	/	28	/	/	/	/	/
Gestation
Days 0-7	/	/	/	/	/	23	23	21	22	24
Days 7-14	/	/	/	/	/	25	25	24	25	25
Days 14-20	/	/	/	/	/	29	29	29	30	32*
Lactation
Days 1-5	/	/	/	/	/	39	40	39	39	36
Days 5-12 (treatment free)	/	/	/	/	/	77	/	/	/	71
Days 12-19 (treatment free)	/	/	/	/	/	99	/	/	/	87
* p < 0.05, ** p < 0.01

 

Table 4: Mean organ weight changes (g)

Sex	Male				Female
Dose level (mg/kg bw/day)	100	250	500	1000	100	250	500	1000
Examined/Number of animals	10/10	10/10	10/10	10/10	10/10	10/10	10/10	10/10
Final bodyweight	+5	+3	+1	+2	-2	-5	-4	-3
-Liver
Absolute	+10*	+10*	+7	+13**	+1	+4	+6	+6
Relative	+5	+7*	+7*	+11**	+3	+10*	+11**	+9
-Kidneys
Absolute	+23**	+24**	+22**	+34**	+6	+5	+6	+2
Relative	+18*	+20**	+22**	+31**	+7	+10*	+10**	+5
* p < 0.05, ** p < 0.01

 

Table 5: Mean organ weight changes (g) treatment-free

Sex	Male	Female
Dose level (mg/kg bw/day)	1000	1000
Examined/Number of animals	5/5	5/5
Final bodyweight	-4	-2
-Kidneys
Absolute	+12	-1
Relative	+17*	+1
* p < 0.05

 

Table 6: Parental animals - gross findings at necropsy

Sex	Males
Dose-level (mg/kg bw/day)	0	100	250	500	1000
Number of animals	10	10	10	10	10
Kidney, irregular colour	0	0	1	6	3
kindey tan discolouration	0	6	9	4	7
liver, accentuated lobular pattern	3	6	7	7	9
liver, tan discolouration	0	2	1	0	1

 

Table 7: Parental microscopic findings

Sex	Male
Dose-level (mg/kg bw/day)	0	100	250	500	1000
Number of animals	10	10	10	10	10
Kidney; degeneration/necrosis; tubule (grade 1 minimal)	0	0	0	0	2
Kidney; basophilia; tubule (grade 1 minimal)	5	2	0	0	0
Kidney; basophilia; tubule (grade 2 slight)	1	4	1	1	1
Kidney; basophilia; tubule (grade 3 moderate)	0	4	9	9	9
Kidney; dilation; tubule (grade 1 minimal)	0	5	7	9	4
Kidney; dilation; tubule (grade 2 slight)	0	3	0	1	6
Kidney; dilated tubules with eosinophilic content (grade 1 minimal)	0	0	4	1	0
Kidney; dilated tubules with eosinophilic content (grade 2 slight)	0	2	4	4	9
Kidney; dilated tubules with eosinophilic content (grade 3 moderate)	0	2	1	4	1
Kidney; hyaline droplets; tubular epithelium (grade 1 minimal)	0	1	0	0	0
Kidney; hyaline droplets; tubular epithelium (grade 2 slight)	0	3	0	1	0
Kidney; hyaline droplets; tubular epithelium (grade 3 moderate)	0	5	9	3	5
Kidney; hyaline droplets; tubular epithelium (grade 4 marked)	0	1	1	6	5
Kidney; Infiltrate; mononuclear cell infiltrate (grade 1 minimal)	1	5	2	1	7
Liver; hypertrophy; hepatocellular (grade 1 minimal)	0	1	4	5	6
Liver; vacuolation; periportal (grade 1 minimal)	2	7	6	4	7
Liver; vacuolation; periportal (grade 2 slight)	0	0	1	3	1

 

Table 8: Parental microscopic findings (treatment-free period)

Sex	Male
Dose-level (mg/kg bw/day)	0	1000
Number of animals	10	10
Kidney; basophilia; tubule (grade 1 minimal)	3	0
Kidney; basophilia; tubule (grade 2 slight)	0	2
Kidney; basophilia; tubule (grade 3 moderate)	0	2
Kidney; basophilia; tubule (grade 4 marked)	0	1
Kidney; dilation; tubule (grade 1 minimal)	1	1
Kidney; dilation; tubule (grade 2 slight)	0	1
Kidney; dilated tubules with eosinophilic content (grade 2 slight)	0	3
Kidney; dilated tubules with eosinophilic content (grade 3 moderate)	0	2
Kidney; hyaline droplets; tubular epithelium (grade 1 minimal)	0	1
Kidney; Infiltrate; mononuclear cell infiltrate (grade 1 minimal)	1	3

 

Table 9: Reproductive and delivery data

Dose level (mg/kg bw/day)	0	100	250	500	1000
Number of animals paired (M+F)	15+15	10+10	10+10	10+10	15+15
Number of males mated	15	10	10	10	15
Number of females mated	15	10	10	10	15
Mating index (%)	100	100	100	100	100
Mean number of days taken to mate	2.2	3.5	2.2	4.2	2.5
Number of pregnant females	13	9	10	10	15
Fertility index (%)	86.7	90	100	100	100
Number of females with live born pups	11	9	10	10	15
Gestation index (%)	84.6	100	100	100	100
Number of females which delivered	11	9	10	10	15
Mean duration of gestation (days)	22	22	21.9	22	21.9
Mean number of corpora lutea	15	15.6	15.6	18.3	16.5
Mean number of implantations	15	15.6	15.6	16	15.5
Mean pre-implantation loss (%)	13.3	0	0	5.8	0.8
Mean number of pups delivered	12.2	12.8	14.5	13.4	13.3
Mean post-implantation loss (%)	17.6	18	6.4	15.6	13.3

 

Table 10: Pup viability indices and sex ratio

Dose level (mg/kg bw/day)	0	100	250	500	1000
Viability index on Day 4 p.p. (%)	97.8	95.7	91	96.3	93
No. of pups cannibalized	0	1	3	1	10
No. of pups found dead	4	4	10	4	4
Sex ratio at birth (% males)	46.3	53.5	62.8	47	50

 

Table 11:Pups Bodyweight (g)

Sex	Male					Female
Dose level (mg/kg bw/day)	0	100	250	500	1000	0	100	250	500	1000
Body weight
Day 1 p.p.	8.2	7.8	7.3	7.7	7.7	7.7	7.3	6.9	7.3	7.4
Day 5 p.p.	14.1	13.8	12.7	12.9	13	13.6	13.3	12.1	12.2	12.5
Day 12 p.p. (treatment free)	26.2	/	/	/	29.2	25.6	/	/	/	26.2
Day 19 p.p. (treatment free)	42.8	/	/	/	48.7	41.2	/	/	/	46.5
Body weight change
Days 1-5 p.p.	6	6	5.4	5.2	5.3	5.9	5.9	5.2	5	5.2
Days 5-19 p.p. (treatment free)	30	/	/	/	35.6	29	/	/	/	33.9
Days 1-19 p.p.	35.2	/	/	/	41.3	33.9	/	/	/	39.4

 

 

Table 12:Pup clinical observations

Dose level (mg/kg bw/day)	0	100	250	500	1000
Haematoma on head	1 (1)				2 (1)
scab on back				1 (1)
wound on urogenital area					2 (1)
Dehydration					1 (1)
Thin appearance					1 (1)
Difficult breathing					1 (1)
Cold to the touch					1 (1)

 

Table 13:Pup macroscopic observations

Dose (mg/kg bw/day)	0	100	250	500	1000
Number of pup (litter) examined	130 (11)	110 (9)	132 (10)	129 (10)	186 (15)
Pale liver		1 (1)	1 (1)
Dilated renal pelvis		1 (1)
Absent kidney and ureter	1 (1) (treatment-free period)

Applicant's summary and conclusion

Conclusions:

The No Observed Adverse Effect Level (NOAEL) for parental systemic toxicity was considered to be 500 mg/kg bw/day for males based on the adverse microscopic effects in kidneys seen at the higher dose and 1000 mg/kg bw/day for females based on the absence of adverse effects in females. However, the renal effects seen in male rats are not relevant to human exposure. The No Observed Effect Level (NOEL) for parental reproductive performance was considered to be 1000 mg/kg bw/day based on the absence of effects in mean reproductive data at all dose levels. The No Observed Adverse Effect Level (NOAEL) for toxic effects on progency was considered to be 1000 mg/kg bw/day based on absence of adverse effects in pups at all dose levels.

Executive summary:

GUIDELINE

The study design was based on OECD 421 of 27 July 1995. The objective was to evaluate the portential toxic effects of the test item following daily oral (gavage) administration to male and female rats from before mating, through mating and, for females, through gestation until day 5 post-partum. On completion of the treatment period, additional dams and litters were held for a two week treatment-free period in the control and high dose groups in order to evaluate the reversibility of any findings. The study provided initial information on male and female reproductive performance, such as gonadal function, mating behaviour, conception, development of the conceptus and partrutition, and on reversibility of potential findings on development of the offspring or gonadal weight and morphology.

METHODS

Four groups of 10 male and 1o female (groups 2 to 4) or 15 male and 15 female (group 5) Sprague-Dawley rats received the test item daily by oral (gavage) administration before mating, through mating and, for females, through gestation until day 5 post-partum. The test item was administered as a solution in the vehicle, corn oil, at dose levels of 100, 250, 500 and 1000 mg/kg bw/day. Another group of 15 males and 15 females received the vehicle alone, under the same experimental conditions, and acted as the control group. A constant dose volume of 4 mL/kg bw/day was used. On completion of the treatment period, the animals in each group were sacrificed, except for five animals per sex in the control and high dose groups which were kept for a 2 -week recovery period. The concentrations of the dose formulations were checked in study weeks 1, 3 and 6.

The animals were checked twice daily for mortality and morbidity and once daily for clinical signs during the treatment and treatment-free periods. Body weight and food consumption were recorded once a week during pre-mating and mating periods (food consumption not during mating), and during gestation on days 0, 7, 14 and 20 post-coitum plus lactation on days 1 and 5 post-partum. These data were also recorded on days 12 and 19 post-coitum for treatment-free females and weekly after the mating period for treatment-free males.

Animals were paired for mating after 2 weeks of treatment and the females were allowed to litter and rear their progeny until day 5 post-partum or until day 19 post-partum for treatment-free females. The total litter sizes and number of pups of each sex were recorded after birth. Pups were observed daily for clinical signs, abnormal behaviour and external abnormalities and weighed on days 1 and 5 post-partum. Pups of treatment-free females were also weighed on days 12 and 19 post-partum.

Males were sacrificed after at least 5 weeks of treatment and females on day 6 post-partum except for treatment-free animals, which were sacrificed 2 weeks after the end of treatment (thus on day 20 post-partum for females). Final body weights and selected organ weights (epididymides, testes, kidneys, liver, spleen) were recorded and a macroscopic post-mortem examination of the prinicpal thoracic and abdominal organs was performed. Particular attention was paid to the reproductive organs. A microscopic examination was performed on ovaries(with oviducts) from all females sacrificed on day 6 post-partum in the control and high dose groups and epididymides and/or testes from all males sacrificed at the end of the treatment period in the control and high dose groups. A microscopic examination was also performed on the kidneys and liver from all males sacrificed at the end of the treatment or treatment-free period in all groups and on all macroscopic lesions. Pups were sacrificed on day 5 post-partum (or day 19 post-coitum for treatment-free pups) and submitted for post-mortem examination of the principal thoracic and abdominal organs.

RESULTS

Chemical analysis: No test item was detectable above the limit of quantification in the control dose formulation and the test item concentrations in the analysed dose formulations were within an acceptable range.

F0 animals: The following findings were reported:

- No premature deaths in the test-item treated groups during treatment and treatment-free periods.

- No toxicologically significant clinical signs during treatment and treatment-free periods.

- No toxicologically significant effects on mean body weights, mean body weight changes and mean food consumption during treatment and treatment-free periods.

- No effects on mean pairing, mating, fertility and delivery data.

- Test item-related kidney and liver mean weight increases were seen in males treated with 100 mg/kg bw/day or above.

- Test item-related irregular colour and or tan discolouration of kidneys and increased incidence of accentuated lobular pattern together with tan discolouration was observed in males treated with 100 mg/kg bw/day or above. At the end of the treatment-free period, there were no test item-related gross findings in either the liver or the kidneys.

- A series of test-item related microscopic findings was found in the kidneys and liver of males treated with 100 mg/kg bw/day or above. These findings were degradation/necrosis of tubules (adverse) in 2/10 males treated at 1000 mg/kg bw/day, hyaline droplets in tubular epithelium, tubular dilation with or without eosinophilic content in kidneys, and hepatocellular hypertrophy plus increased periportal vacuolation in the liver. At the end of the treatment-free period, the reversibility of the liver changes was complete whilst reversibility of kidney changes was incomplete.

Pups: The following findings were reported:

- No test item-related effects on pup viability during treatment and treatment-free periods.

- No test-item related clinical signs during treatment and treatment-free periods.

- No toxicologically significant effects on mean body weight and mean body weight gains during treatment and treatment-free periods.

- No clear indication of a test item treatment effect on percentage of male pups at birth.

- No test item-related necropsy findings during treatment and treatment-free periods.

CONCLUSION

The No Observed Adverse Effect Level (NOAEL) for parental systemic toxicity was considered to be 500 mg/kg bw/day for males based on the adverse microscopic effects in kidneys seen at the higher dose and 1000 mg/kg bw/day for females based on the absence of adverse effects in females. However, the renal effects seen in male rats are not relevant to human exposure. The No Observed Effect Level (NOEL) for parental reproductive performance was considered to be 1000 mg/kg bw/day based on the absence of effects in mean reproductive data at all dose levels. The No Observed Adverse Effect Level (NOAEL) for toxic effects on progency was considered to be 1000 mg/kg bw/day based on absence of adverse effects in pups at all dose levels.