Methoxycarbonyloxycyclooct-4-ene

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Remarks:

TNO Triskelion, Utrechtseweg 48, 3704 HE Zeist, The Netherlands

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Methoxycarbonyloxycyclooct-4-ene

EC Number:

401-620-8

EC Name:

Methoxycarbonyloxycyclooct-4-ene

Cas Number:

87731-18-8

Molecular formula:

C10H16O3

IUPAC Name:

(1R,3aS,6aS)-octahydropentalen-1-yl methyl carbonate; (1S,3aS,6aS)-octahydropentalen-1-yl methyl carbonate; (3Z)-cyclooct-3-en-1-yl methyl carbonate; (4Z)-cyclooct-4-en-1-yl methyl carbonate; bicyclo[3.2.1]octan-8-yl methyl carbonate

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan, Horst, the Netherlands.
- Age at study initiation: females approximately 8 weeks and males approximately 9 weeks
- Weight at study initiation: ranged from 297 to 334 g for the males and from 171 to 202 g for the females.
- Housing: Animals were housed in macrolon cages with a bedding of wood shavings (Lignocel) and strips of paper (Enviro-dri) and a wooden block
as environmental enrichment. During the premating period, the animals were, as far as possible, housed in groups of 4/sex. For mating, one male and one female were housed together. Mated females were housed individually in macrolon cages.
- Diet: Cereal-based (closed formula) rodent diet (VRF-1 (FG)) from a commercial supplier (SDS Special Diets Services, Witham, England), ad libitum.
- Water: Tap-water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65%
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

The diets were mixed in a mechanical blender. Two batches of the experimental diets were prepared in the study on 17 October 2014 and on 14 November 2014) and stored in a freezer (<-18°C) in plastic bags in portions sufficient for seven days. The feed was provided as a powder in stainless steel cans, covered by a perforated stainless steel plate to prevent spillage. The feed in the cans was replaced once weekly with fresh portions from the freezer.

Details on mating procedure:

At the end of the premating period, each female was caged with one male from the same group. Animals were caged together until mating was successful. Mating pairs were clearly identified. Every consecutive morning during the mating period, vaginal smears were made for determination of the presence of sperm. The day on which sperm was detected in the vaginal smear was considered as gestation day 0. Upon evidence of copulation the females were caged individually for the birth and rearing of their pups. Dams were allowed to raise their litter until sacrifice on day 4 of lactation or shortly thereafter.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

During the dose-range finding study (non-GLP), an analytical method to measure the concentration of the test substance in the experimental diets was developed. Before analysis of study samples, this analytical method was validated in the current study to conform to the following criteria:
- Linearity: the correlation coefficient of the calibration curve should be greater than or equal to 0.996;
- Recovery: the mean recovery of the test substance from the formulation should be between 80% and 110% at each of the dose levels of the study;
- Repeatability: the relative standard deviation in the percentage recovery, when the recovery test is performed three times at each of the dose levels to be used in the toxicity study, should be less than 10%.
Selectivity: no peak should be found in blank sample with a retention time of 95-105% of that of the test substance. If the blank sample shows a peak with a retention time in the above range and the area of this peak
is >5% of the corresponding peak in the chromatogram from a low-dose sample, the results for the samples containing test substance will be corrected for the level found in blank sample. From the first batch of dosing formulations prepared in the study on 17 October 2014, samples were taken and analyzed. The method of analysis for detection of the test substance in the carrier was reversed phase HPLC and UV-detection. The following analyses were conducted in the first batch of dosing formulations prepared in the study:
- Homogeneity of the test substance at each dose level (5 samples per dose level, taken at different locations in the feed container). The samples taken for homogeneity experiments were also used for dose confirmation on the day of preparation*.
- Stability of the test substance under experimental conditions (one sample per dose level and one control sample, after storage for about 7 days* in the animal room and after storage for at least 5 weeks in a freezer at <-18°C). As a second batch of dosing formulations was prepared in the study on 14 November 2014, samples were taken and content was measured.

*Between sampling and analysis, the samples were temporarily stored in a freezer (<-18°C).

Duration of treatment / exposure:

Males 34 days. Females received substance during premating period of 2 weeks, mating (1 week) gestation and lactation until postnatal day 4

Frequency of treatment:

Continuously

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 500 mg/kg diet

Remarks:

Nominal concentration: 100 mg/kg bw/day
Actual ingested (males): 59 mg/kg bw/day
Actual ingested (females): 78 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

5 000 mg/kg diet

Remarks:

Nominal concentration: 250 mg/kg bw/day
Actual ingested (males): 209 mg/kg bw/day
Actual ingested (females): 252 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

7 500 mg/kg diet

Remarks:

Nominal concentration: 500 mg/kg bw/day
Actual ingested (males): 332 mg/kg bw/day
Actual ingested (females): 378 mg/kg bw/day

No. of animals per sex per dose:

12

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dose levels were selected in consultation with the sponsor and are based on the results of a 2-weeks dose-range finding study with Violiff in rats.

Examinations

Parental animals: Observations and examinations:

- General clinical observations: Each animal was observed daily in the morning hours by cage-side observations. On working days, all cages were checked again in the afternoon for dead or moribund animals to minimize loss of animals from the study. All abnormalities, signs of ill health or reactions to treatment were recorded.
- Body weight: Body weights of male and female animals were recorded shortly before the start of the treatment (at randomization) and at the start of the study (day 0). Subsequently males were weighed weekly until sacrifice. Females were weighed once per week during the premating and mating period. Mated females were weighed on days 0, 7, 14 and 21 during presumed gestation and on day 0 and 4 of lactation. Non-mated females were weighed once per week after the mating period. The animals were weighed on their scheduled necropsy date in order to calculate the correct organ to body weight ratios.
- Food consumption: Food consumption was measured per cage over the same periods as the body weight are measured. Food consumption was not recorded during the mating period. The results are expressed in g per animal per day. Food was refreshed once weekly.

Litter observations:

- Parturition and litter evaluation: At the end of the gestation period (GD 21), females were examined twice daily for signs of parturition. Any difficulties occurring during parturition were recorded. To keep nest disturbance to a minimum the litters were examined only once daily for dead pups.

- Litter size, sexes and weight: The total litter size and numbers of each sex as well as the number of stillbirths, live- and dead pups and grossly malformed pups was evaluated on days 0 and 4 of lactation. The pups were individually weighed on days 0 and 4 of lactation. Mean pup weight was calculated per sex and for both sexes combined.

Postmortem examinations (parental animals):

All surviving male and female parent animals were sacrificed by exsanguination from the abdominal aorta whilst under CO2/O2 anaesthesia at necropsy and then examined grossly for pathological changes. Male animals were sacrificed after treatment for 34 days. Female animals were sacrificed at or shortly after day 4 of lactation. Samples of the following tissues and organs of all parent animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde; except for the testes which were preserved in Bouin's fixative: ovaries (after counting the corpora lutea), uterus (after counting of the implantation sites), testes, epididymides, seminal vesicles, prostate, kidneys, liver, all gross lesions. The testis, epididymides, kidneys and liver were weighed (paired organs together) as soon as possible after dissection to avoid drying. Tissues for microscopic examination were embedded in paraffin wax, sectioned at 5 µm, and stained with haematoxylin and eosin, except for sections of the testes which were stained with PAS haematoxylin. Microscopic examination was performed on the collected ovaries, uterus, testes, epididymides, seminal vesicles and prostate of all animals of the control and high concentration group. The prostate and seminal vesicles from male animal 86 (high dose group) were lost during necropsy and were not examined histopathologically. Livers and kidneys of all male animals were microscopically examined. Livers were stained with haematoxylin and eosin. The kidneys of the males were analysed for α2-globulin accumulation using histochemistry. Furthermore, organs showing gross lesions of animals of all groups were examined microscopically.

Postmortem examinations (offspring):

No pups were malformed or stillborn and subjected to macroscopic examination. At necropsy of the dams, at or shortly after day 4 of lactation, pups were examined externally for gross abnormalities and killed by appropriate techniques. Pups were stored in a freezer for possible skeletal analyses until finalisation of the report, thereafter they will be discarded.

Statistics:

The data were analysed using appropriate methods. Tests were generally performed as two-sided tests with results taken as significant where the probability of the results is p<0.05 or p<0.01. Continuous data were subjected to the “Decision tree for continuous data” and dichotomous data were subjected to the “Decision tree for dichotomous data”.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical signs observed were restricted to skin observations and were not considered to be treatment related (see Table 1 in the attached report, starting at page 26).

Mortality:

no mortality observed

Description (incidence):

No mortality and morbidity was observed during premating, mating, gestation and lactation (see Table 1 in the attached report, starting from page 26).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Both males and females of the high dose group show a statistically significantly decreased body weight gain during the first week of the treatment period. This was considered to be related to the palatability of the test substance. For males no further effects on body weight or body weight gain were observed. For females no statistically significant effects were observed on bodyweight during premating. During lactation a slightly, but statistically significantly lower body weight was observed in the females of the high dose on lactation day 4. Taking into account that the effect is minimal (6%) and that relative the body weight gain during the first four days of lactation is comparable with the control animals, this is not considered of toxicological relevance (see Tables 2 and 3 in the attached report, from page 33 onwards).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean food consumption was statistically decreased during the first week of treatment for males and females in both the mid dose group and the high dose group and recovered thereafter. This is considered to be related to the palatability of the test substance. During gestation food consumption was comparable in all groups. However, during lactation a statistically significantly lower food consumption was observed in the high dose females. This was considered to be related to treatment.

The mean test substance intake was calculated based on measured food consumption and body weight data. In view of the instability of the test item in diet at ambient temperature in the animal room for 7 days, actual test substance intake was corrected for the instability. Assuming that the majority of the test substance disappeared already within the first day of use, a worst case calculation was made, resulting in calculated test substance intakes of at least 59 mg/kg body weight for males and 78 mg/kg body weight for females in the low dose group, at least 209 mg/kg body weight for males and 252 mg/kg body weight for females in the mid dose group and at least 332 mg/kg body weight for males and 378 mg/kg body weight for females in the high dose group, (see Tables 4 in the attached report, page 43 onwards)

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic examination of the liver revealed no treatment-related abnormalities. In the kidney a dose dependent increase of hyaline droplets in the proximal tubular epithelium cells of the kidneys of all males of the lowdose, mid-dose and high-dose groups were observed. Most males also showed basophilic tubules in the kidneys, some males showed dilated tubules, filled with eosinophilic debris.
These histological observations are in agreement with the description of nephrotoxicity induced by the accumulation of α2 microglobulin, characterized by enlarged lysozomes (hyaline droplets), tubule dilatation as a result of the exfoliation of the proximal tubule epithelium and the accumulation of tubular cell debris (dilated tubules filled with eosinophilic debris) and cell tubular proliferation (basophilic tubules). The presence of α2-microglobulin was confirmed by immunohistochemical staining (see Table 12 in the attached report, page 63).

Reproductive function / performance (P0)

Reproductive performance:

no effects observed

Description (incidence and severity):

Each group comprised 12 males and 12 females and all mating pairs were successfully mated and shown pregnant, resulting in fertility and gestation indices of 100%.The mean number of mating days until successful copulation was comparable in all groups. No effects on male or female fertility were observed. All females in all groups delivered litters with liveborn pups. No stillborn pups were delivered. The mean number of gestation days was comparable in all groups. The mean number of corpora lutea, mean number of implantation sites and the mean number of pups delivered in the control group was slightly higher than in the treatment groups. A total of 150, 137, 135 and 136 pups were born in the control group, low dose, mid dose and high dose groups, respectively. In absence of a statistically significantly difference and in absence of a dose response relationship this is considered to be a chance finding. No effects on reproductive performance were observed (see Table 5, 6 and 7 in the attached report, starting at page 52).

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

parental toxicity

Effect level:

ca. 332 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: corresponding to NOAEL ≥ 7500 mg test substance/kg diet

2

Dose descriptor:

NOAEL

Remarks:

parental toxicity

Effect level:

ca. 378 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: corresponding to NOAEL ≥ 7500 mg test substance/kg diet

3

Key result

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

ca. 332 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: corresponding to NOAEL ≥ 7500 mg test substance/kg diet

4

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

ca. 378 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: corresponding to NOAEL ≥ 7500 mg test substance/kg diet

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

One male pup in the control group showed a subcutaneous haemorrhage on the head. No other clinical signs were observed (see Table 8 of the attached report, page 57)

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Pup survival until postnatal day 4 (Alive Day 4) was 98.7, 98.5, 99.3 and 97.1% in the control, low dose, mid dose and high dose group, respectively (see Table 7 of the attached report, page 56)

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean pup weight was comparable on postnatal days 0 and 4. No effects on pups weight were observed (see Table 9 of the attached report, page 59).

Gross pathological findings:

no effects observed

Description (incidence and severity):

All pups that did not survive until postnatal day 4 were missing in the litter. It was concluded that these pups had been cannibalized and therefore macroscopic examination was not possible.

Description (incidence and severity):

Pup sex was comparable in all groups (50.7% males in the control group on lactation day 0 and 48.9%, 47.4% and 49.3% for the low dose, mid dose and high dose groups, respectively).

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 332 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed up to and including highest dose level

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Analytical results

The analytical report concludes that:

- The results of the homogeneity analyses showed that Violiff was homogeneously distributed in the diets.

- The results of the stability experiments showed that Violiff was not considered to be stable in the diets at ambient temperature in the animal room. A relative decrease of the Violiff concentration was 29 % for the low dose group, 24% for the mid dose group and 22% for the high dose group. Upon storage in the freezer (at < -18 ºC) in a closed container for 5 weeks, the relative decrease of the Violiff concentration was less than 10% for the high dose level. With relative differences upon torage for 5 weeks in the freezer of 15% and 12 % for the low – and mid-dose level respectively, Violiff was not stable in diet at < -18 ºC in a closed container for 5 weeks.

- The results of the test item content analysis of the diets prepared on 17 October 2015 and 14 November 2014 showed that the concentration of the test item was close to intended (relative difference < 10%) for all dose levels except for the low level diets made on 15 November 2014 (- 11% instead of < 10% relative difference).

- Stability analyses in the main study, using a validated method, showed the following data after storage:

Nominal levels of VIOLIFF	Relative difference with initial analysis, upon storage in the animal room for 7 days* (%)	Relative difference with nominal level upon storage in the animal room for 7 days* (%)
1500 mg/kg diet	-29%	-27%
5000 mg/kg diet	-24%	-20%
7500 mg/kg diet	-22%	-19%

* The result of analysis also showed some loss after storage for 5 weeks in the freezer. However, because the above samples (after storage in the animal room) were stored in the freezer for several weeks until analysis, this loss is already included in the above figures.

 

Assuming that the majority of the test substance disappeared already within one day of use, the stability figures in column 3 were used for a worst case calculation of the daily actual intake of the test substance.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the test (OECD 421, GLP), the NOAEL for parental toxicity and fertility were determined to be 332 and 378 mg/kg bw for males and females, respectively. The NOAELs were based on the absence of toxicological relevant adverse effects at a dose of 7500 mg substance/kg diet.

Executive summary:

In an OECD 421 guideline study performed in compliance with GLP, male and female Wistar rats were exposed to the test substance via the diet at nominal mg/kg bw doses of 100 (1500 ppm), 300 (5000 ppm) or 500 (7500 ppm). The doses in the diet were selected based on the 14-day DRF. Exposure to the substance covered a premating period of 2 weeks, during mating (1 week), gestation and lactation until postnatal day 4. In life parameters included clinical observations, body weight, and food consumption. At necropsy animals were macroscopically examined, liver and kidney weight were recorded. Male (testes, epididymides, seminal vesicles and prostate) and female (ovaries and uterus) reproductive organs, kidneys and livers were examined microscopically.

Results: Analysis: The test item was considered to be homogeneously distributed in the diets. The results of the stability experiments showed that the test item was not stable in the diets at ambient temperature in the animal room. Relative decreased of the test item concentration were 29%, 24% and 22% after seven days for the low dose, mid dose and high dose groups, respectively. Analysis for test item content of the diets showed that the concentration of the test item in the diets was close to intended.

Clinical signs: No mortality or morbidity was observed during the study. Statistically significantly decreased body weight gains during the first week of treatment in males and females in the high dose group was seen. This was accompanied with statistically significantly reduced food consumption during the first week of treatment in males and females in the mid dose and high dose group as compared to the control group. These effects were related to palatability of the test substance and not considered adverse.

Organ weights: Statistically significantly increased mean relative liver weight in males of the high and mid dose: +23 and +15%, respectively not accompanied by microscopic abnormalities. Statistically significantly dose related increased relative kidney weights in males of all test item-treated groups were observed (+16% in the high dose. These were related to alpha-2u-globulin nephropathy (confirmed by immune staining) and therefore not considered adverse for humans. No effects in females were seen on these organs.

Fertility: Finally, no effects on organ weight, macroscopical or microscopical examination of the reproductive organs were observed. No effects on mating index, fertility index, pregnancy rates, gestation index or days. No effects on number of corpora lutea, implantation sites or preimplantation loss.

Developmental toxicity: No effects on total number of, live born or still born pups. No effects on prenatal loss or sex ratio. No effects on pup weights up to day 4.

Conclusion: Based on the absence of adverse effects in males and females the NOAEL for parental toxicity was established at 500 mg/kg bw (nominal) corresponding to a dose of at least 332 mg/kg body weight per day for males and at least 378 mg/kg body weight for females.