2,4,8,10-Tetraoxaspiro[5.5]undecane-3,9-diethanol, .beta.3,.beta.3,.beta.9,.beta.9-tetramethyl-

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 27 June 2013 and 22 August 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

See discussion

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Rat, RccHan: WIST(SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst, Netherlands.
- Age at study initiation: 11 weeks
- Weight at study initiation: Males: 349 to 401 g; females: 200 to 224 g
- Housing: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood with paper enrichment. During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Use of restrainers for preventing ingestion (if dermal): Not applicable
- Diet (e.g. ad libitum): Pelleted standard Harlan Teklad 2018C (batch no. 56/12) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst, Switzerland) was available ad libitum.
- Water (e.g. ad libitum): Community tap-water from Itingen was available ad libitum in water bottles.
- Acclimation period: At least five days of acclimatization under test conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour fluorescent light / 12-hour dark cycle with at least eight hours music during the light period.

Route of administration:

oral: gavage

Vehicle:

peanut oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared weekly using the test item as supplied by the Sponsor.
Spiroglycol (SPG) was weighed into a glass beaker on a tared precision balance and the vehicle was added. Using an appropriate homogenizer, a homogeneous suspension was prepared. Separate formulations were prepared for each concentration. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

Storage of Dose Formulations
Dose formulations were stored in glass bottles at room temperature (20 ± 5 ºC) for the first week of treatment and were stored in refrigerator (5 ± 3 °C) thereafter until the end of the study. Based upon the results of stability analyses performed within the non- GLP Harlan Laboratories Ltd. study no. D79374 (Implementation and validation of an analytical method for dose formulation analysis), dose formulations were stable for 4 hours at room temperature and for 8 days in the refrigerator.


VEHICLE
- Amount of vehicle (if gavage): Dose volume: 5 mL/kg body weight
- Lot/batch no.: MKBK9131V

Details on mating procedure:

During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed. The females were removed and housed individually if:
- the daily vaginal smear was sperm positive, or
- a copulation plug was observed.
The day on which a positive mating was determined (copulation plug or sperm) was designated day 0 post coitum. If a female did not mate during the 14-day pairing period, a second pairing of this female with a male in the same group, which had already mated successfully, was considered. All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 1 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 1 g of each concentration were taken from the middle to confirm the stability (4 hrs at room temperature and 8 days in refrigerator and 8 days at room temperature). During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were delivered to the responsible person for formulation analysis and stored there at -20 ± 5 °C until analysis.
The samples were analyzed by gas chromatography (GC) method following an analytical procedure provided by the Sponsor and adapted at Harlan Laboratories Ltd. The test item was used as the analytical standard. Analyzed samples were not discarded without written consent from the study director.
Duplicates were taken of all samples and were stored at Harlan Laboratories Ltd., Itingen, Switzerland. The samples were not discarded without written consent from the study director.

Duration of treatment / exposure:

Males: Minimum 4 weeks
Females: Approximately 7 weeks

Frequency of treatment:

Daily at approximately 24 hour intervals.

Details on study schedule:

Acclimatization: 5 days minimum for males and females.
First Test Item Administration: Day 1 of pre-pairing for males and females.
Pre-Pairing: 14 days for males and females.
Pairing: 14 days for males and females.
Gestation Approximately 21 days
Treatment Ends On day before sacrifice for males; on day 4 post partum for females
Necropsy After a minimum of 43 days treatment (on Day 47 of the study) for males; on day 5 post partum for females.

Remarks:

Doses / Concentrations:
0 mg/kg bw/day (control group), 30 mg/kg bw/day, 300 mg/kg bw/day, 1000 mg/kg bw/day, equivalent to 0 mg/mL, 6 mg/mL, 60 mg/mL and 200 mg/mL.
Basis:
actual ingested

No. of animals per sex per dose:

12/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a 28-day toxicity study in Han Wistar rats, (Harlan Laboratories UK study no. 0930-0125), using dose levels of 0, 15, 150 and 1000 mg/kg bw/day, resulting in a NOEL (no-observed-effect-level) of 1000 mg/kg bw/day.
- Rationale for animal assignment (if not random): Randomisation was performed after at least three days of acclimatization using a computer-generated random algorithm. Body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.

Parental animals: Observations and examinations:

Viability / Mortality: Twice daily

Clinical Signs: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

Food Consumption:
Males: Weekly during pre-pairing and after pairing periods.
Females: Pre-Pairing period days 1 - 8 and 8 - 14, gestation period days 0 - 7, 7 - 14 and 14 - 21 post coitum and lactation period days 1 - 4 post partum.
No food consumption was recorded during the pairing period.

Body Weights: Recorded daily from treatment start to day of necropsy.

Litter observations:

The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post partum.

Postmortem examinations (parental animals):

Males were sacrificed when they were no longer needed for the assessment of reproductive effects (on day 47 of the study). Dams and pups were sacrificed on day 5 post partum. If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.

At the scheduled sacrifice, all animals were weighed and sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated. All parent animals, except those excessively cannibalized, were examined macroscopically for any structural changes at the scheduled necropsy. For the parent animals, special attention was directed at the organs of the reproductive system. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4 % formaldehyde solution. The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

Organ Weights and Preservation
At the scheduled sacrifice, organs were weighted and preserved. Organs were trimmed from any adherent tissue, and their wet weight taken. If not indicated otherwise, organs were preserved in neutral phosphate buffered 4 % formaldehyde solution.

The following tissues were collected and examined:
Epididymides (fixed in modified Davidson's solution)*
Ovaries
Uterus (incl. oviducts, cervix and vagina)
Mammary gland
Prostate gland and seminal vesicles incl. coagulating glands
Testes (fixed in modified Davidson's solution)*
Pituitary
All gross lesions

The following tissues were also weighed:
Epididymides (fixed in modified Davidson's solution)*
Testes (fixed in modified Davidson's solution)*
Pituitary
* Testes and epididymides were weighed separately.

Histotechnique
All organ and tissue samples to be examined by the principal investigator were processed, embedded and cut at an approximate thickness of 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis was stained with PAS. Special stains were used at the discretion of the study pathologist.

Histopathology
Slides of all organs and tissues collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the principal investigator. The same applied to all occurring gross lesions.
Qualitative assessment of the male reproductive organs was performed. Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.
Histological examination of all organs was carried out on the organs listed above for any females that did not give birth and all infertile males of all dose groups.
A histopathology peer review was performed.

Postmortem examinations (offspring):

All pups, except those excessively cannibalized, were examined macroscopically for any structural changes at the scheduled necropsy.

Statistics:

For reproduction data, group mean values were calculated both on a litter basis and on a percentage per group basis. Mean pup weights were calculated from the individual weights both on a per group and on a per litter basis.

Means and standard deviations of various data were calculated and included in the report.
The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
All animals survived until the scheduled necropsy. No clinical signs were observed during the study.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No effect on food consumption was noted in males in any dose group. Food consumption of females was not affected by the treatment with the test item.
Body weight of males was not affected by the treatment with the test item. At 1000 mg/kg/day, the body weight gains were statistically significantly increased for a few days during pre-pairing and after pairing periods and the difference in body weights were statistically significantly increased between days 1 and 8 in the pre-pairing period but these were considered not to be test item-related.

Pre-Pairing, Pairing, Gestation and Early Lactation Periods:
No test item-related effects on body weights and body weight gains of females were observed at any dose level.
The body weights of females were statistically significantly increased on days 2 and 5 of the lactation period.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Mating Performance and Fertility
No effect on mating performance or fertility was observed at any dose level.
With exception for one female in the control group (no. 58) mating of all females was recorded during the first pairing period. The pre-coital time was not affected by the treatment with the test item. Mean (median) pre-coital times calculated for the first pairing period were 3.3 (3), 2.7 (3), 3.0 (3) and 2.7 (3) days in order of ascending dose levels. One female in the control group (no. 50), three in group 2 (nos. 61, 63, 66), four females (nos. 73, 76, 77, 79) in group 3 and two females (nos. 86, 88) in group 4 were not pregnant. Fertility indexes (number of females achieving pregnancy as a percentage of females paired) and conception rates (number of females achieving pregnancy as a percentage of females mated) were 91.7 %, 75.0 %, 66.7 % and 83.3 in the control, group 2, group 3 and group 4, respectively.

All pregnant females gave birth to living pups. Gestation index (number of females with living pups as a percentage of females pregnant) was 100% in all dose groups.

Duration of Gestation
No effects on gestation length were observed at any dose level. Mean duration of gestation was 21.6, 21.4, 21.6 and 21.8 days, in order of ascending dose level.

Corpora Lutea Count
No effects on corpora lutea count were observed at any dose level. Mean number of corpora lutea per dam was 14.8, 13.9, 14.0 and 14.1 in order of ascending dose levels.

Implantation Rate and Post-Implantation Loss
No effects on implantations or post-implantation loss were observed. The number of implantations was slightly lower in group 4 but it was not statistically significant and the mean value (11.9) was within the historical background data (10.5 – 14.4).

Litter Size at First Litter Check
No effects on litter size were noted in any dose group at first litter check. The overall mean numbers of living pups per dam at first litter check were 12.7, 11.9, 12.3 and 11.2, whereas birth indexes (number of pups borne alive as a percentage of implantations) were 92.1 %, 92.2 %, 95.1 % and 94.1 % at the dose levels of 0, 30, 300 and 1000 mg/kg bw/day, respectively.

Postnatal Loss Days 0 - 4 Post Partum
No effect on post natal loss was noted between days 0 and 4 post partum. Two pups from group 1 (litter no. 51) were missing and one pup from group 2 (litter no. 70) had spontaneous death on day 2 post partum.


ORGAN WEIGHTS (PARENTAL ANIMALS)
No test item-related finding on organ weights of males or females were noted.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No macroscopical findings were observed at necropsy.

HISTOPATHOLOGY (PARENTAL ANIMALS)

OTHER FINDINGS (PARENTAL ANIMALS)
There were no treatment-related findings.
In particular, qualitative examination of the stages of spermatogenesis in the testis did not reveal any treatment-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle. No treatment-related microscopic abnormalities were observed in the evaluation of the ovarian follicles and corpora lutea of the ovaries.

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest tested dose (limit dose) without effects.

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

CLINICAL SIGNS (OFFSPRING)
No test item-related observations were noted in pups during the first litter check or during the early lactation at any dose level. In the control group one pup had wound on the neck region from day 2 to day 4 post partum. No observations of pups were noted in the test item-treated groups.

BODY WEIGHT (OFFSPRING)
Body Weights to Day 4 Post Partum:
No effects on pup body weights were noted at any dose level. Mean body weights of pups on day 1 post partum were: 6.2 g, 6.3 g, 6.5 g and 6.8 g, at the dose levels of 0, 30, 300 and 1000 mg/kg/day, respectively, body weight gain of pups during the first four days of the lactation period was +44.5%, +47.5%, +49.3% and +52.8%, respectively.

GROSS PATHOLOGY (OFFSPRING)
No test item-related findings were noted at macroscopic examination of F1 pups. For one male pup in control and for one female pup in group 3 the sore skin was recorded.

OTHER FINDINGS (OFFSPRING)
Pups sex ratio was not affected by exposure to the test item at any dose level. At first litter check, percentages of male pups were 51 %, 51 %, 40 % and 50 %, in order of ascending dose level.

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat. (total fraction)

Sex:

male/female

Basis for effect level:

other: highest tested dose (limit dose) without effects.

Reproductive effects observed:

not specified

P Animals Breeding for F1 Litters

Group (mg/kg/day)	1 (0)	2 (30)	3 (300)	4 (1000)
Female numbers	49 – 60	61 – 72	73 – 84	85 – 96
No. of females paired	12	12	12	12
Number of females mated	11	12	12	12
Number of pregnant females (A)	11	9	8	10
Number of non-pregnant females (B)	1	3	4	2
Number of females, which lost their litters	0	0	0	0
Numbers of females, which did not deliver any pups	0	0	0	0
Number of females which reared their pups until day 5post partum	11	9	8	10

(A) Female No. 58 did not mate with male no. 9 in the first pairing period but mated with male no. 10 in the second pairing period.

(B) Female no. 50 in group 1, females 61, 63, 66 in group 2, female nos. 73, 76, 77, 79 in group 3 and female nos. 86, 88 in group 4 were not pregnant.

Conclusions:

No test item-related effects were observed on body weights, body weight gains, food consumption of males and females at the dose level up to and including the dose 1000 mg/kg bw/day. At 1000 mg/kg/day, the body weight gains were statistically significantly increased for a few days during pre-pairing and after pairing periods and the difference in body weights were statistically significantly increased between days 1 and 8 in the pre-pairing period but these were considered of not toxicological relevance.
No effects on mating performance, fertility, corpora lutea count or duration of gestation were observed at any dose level. No effects on implantations or post-implantation loss were observed.
No test item-related findings were noted at first litter check and during lactation in pups up to day 4 post partum at any dose level.
During necropsy and following histopathological examination changes no test item-related findings were noted.
Based on these results, NOEL (No Observed Effect Level) for general toxicity and for reproduction/ developmental toxicity in males and females was considered to be 1000 mg/kg bw/day, the highest does level tested.

Executive summary:

Introduction

The purpose of this study was to generate preliminary information concerning the effects of Spiroglycol (SPG) on male and female reproductive performance such as gonadal function, mating behavior, conception and parturition.

Four groups of 12 males and 12 females were treated by gavage with Spiroglycol (SPG) once daily. Males were treated over a 14-day pre-pairing period and during the pairing period up to one day before necropsy. Females were treated throughout the pre-pairing, pairing, gestation and lactation period up to the day 4post partum.

The following dose levels were used:

Group 1: 0 mg/kg body weight/day (control group)

Group 2 30 mg/kg body weight/day

Group 3: 300 mg/kg body weight/day

Group 4 1000 mg/kg body weight/day

A standard dose volume of 5 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (Peanut Oil).

 

Parent Animals

Mortality and General Tolerability

All animals survived the scheduled study period. No test item-related clinical signs were noted at any dose level.

Food Consumption

Food consumption was not affected by the treatment with the test item at any dose level.

Body Weights

Body weights and body weight gains of males and females were not affected by the treatment up to and including the dose level of 1000 mg/kg body weight/day.

Reproduction and Breeding Data

No effects on mating performance, fertility, corpora lutea count, duration of gestation, post-implantation loss, litter size or breeding loss were observed at any dose level.

Organ Weights

No effects on measured organ weights were noted in males and females.

Macroscopic / Microscopic Findings

There were no treatment-related findings.

 

Litter Data - F1 Pups

Findings at First Litter Check and during Lactation

No test item-related findings were noted in pups at any dose level. Pups sex ratio was not affected by the exposure to the test item at any dose level.

Pup Weights to Day 4Post Partum

No effects on pup body weights or body weight gain were noted at any dose level.

Macroscopic Findings

At necropsy of pups, there were no abnormal findings.

Microscopic Findings

There were no treatment-related findings.

Conclusion

Based on these results, NOEL (No Observed Effect Level) for general toxicity and for reproduction/ developmental toxicity in males and females was considered to be 1000 mg/kg bw/day, the highest does level tested.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The purpose of the reproduction/developmental toxicity screening study was to generate preliminary information concerning the effects of the test substance on male and female reproductive performance such as gonadal function, mating behaviour, conception and parturition. The test item was administered once daily orally (by gavage) to male and female rats throughout the pre-pairing and pairing periods, after pairing (males), gestation and early lactation periods (females) including the day before scheduled necropsy.

 

The study is a valid investigation of the toxicological effects resulting from repeated oral gavage administration of the test item to rats. The test substance was administered in peanut oil as vehicle at dosages of 30, 300, and 1000 mg/kg body weight/day, and controls received the vehicle only. The test substance was administered to male rats for 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

 

No test item-related effects were observed on body weights, body weight gains, food consumption of males and females at the dose level up to and including the dose 1000 mg/kg bw/day. At 1000 mg/kg/day, the body weight gains were statistically significantly increased for a few days during pre-pairing and after pairing periods and the difference in body weights were statistically significantly increased between days 1 and 8 in the pre-pairing period but these were considered of not toxicological relevance.

 

No effects on mating performance, fertility,corpora luteacount or duration of gestation were observed at any dose level. No effects on implantations or post-implantation loss were observed.

 

During necropsy and following histopathological examination changes no test item-related findings were noted.

Based on these results, NOEL (No Observed Effect Level) for general toxicity and for reproduction/ developmental toxicity in males and females was considered to be 1000 mg/kg bw/day, the highest does level tested.

Short description of key information:
A reproduction/developmental toxicity screening test was performed according to OECD Guideline 421. The NOEL (No Observed Effect Level) for general toxicity and for reproduction/ developmental toxicity in males and females was considered to be 1000 mg/kg bw/day, the highest does level tested.

Justification for selection of Effect on fertility via oral route:
A reproduction/developmental toxicity screening test was performed to assess the effects of the test substance on male and female reproductive performance such as gonadal function, mating behaviour, conception and parturition. The test was performed to GLP according to OECD Guideline 421 in an appropriate test species.

Effects on developmental toxicity

Description of key information

A reproduction/developmental toxicity screening test was performed according to OECD Guideline 421. The NOEL (No Observed Effect Level) for general toxicity and for reproduction/ developmental toxicity in males and females was considered to be 1000 mg/kg bw/day, the highest does level tested. 

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 27 June 2013 and 22 August 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

other: OECD guideline 421 Reproduction/Developmental Toxicity Screening Test

Deviations:

yes

Remarks:

See discussion

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: RccHan: WIST(SPF)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst, Netherlands.
- Age at study initiation: 11 weeks
- Weight at study initiation: Males: 349 to 401 g; females: 200 to 224 g
- Housing: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood with paper enrichment. During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Use of restrainers for preventing ingestion (if dermal): Not applicable
- Diet (e.g. ad libitum): Pelleted standard Harlan Teklad 2018C (batch no. 56/12) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst, Switzerland) was available ad libitum.
- Water (e.g. ad libitum): Community tap-water from Itingen was available ad libitum in water bottles.
- Acclimation period: At least five days of acclimatization under test conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour fluorescent light / 12-hour dark cycle with at least eight hours music during the light period.

Route of administration:

oral: gavage

Vehicle:

peanut oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared weekly using the test item as supplied by the Sponsor.
Spiroglycol (SPG) was weighed into a glass beaker on a tared precision balance and the vehicle was added. Using an appropriate homogenizer, a homogeneous suspension was prepared. Separate formulations were prepared for each concentration. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

Storage of Dose Formulations
Dose formulations were stored in glass bottles at room temperature (20 ± 5 ºC) for the first week of treatment and were stored in refrigerator (5 ± 3 °C) thereafter until the end of the study. Based upon the results of stability analyses performed within the non- GLP Harlan Laboratories Ltd. study no. D79374 (Implementation and validation of an analytical method for dose formulation analysis), dose formulations were stable for 4 hours at room temperature and for 8 days in the refrigerator.


VEHICLE
- Amount of vehicle (if gavage): Dose volume: 5 mL/kg body weight
- Lot/batch no. (if required): MKBK9131V

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 1 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 1 g of each concentration were taken from the middle to confirm the stability (4 hrs at room temperature and 8 days in refrigerator and 8 days at room temperature). During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were delivered to the responsible person for formulation analysis and stored there at -20 ± 5 °C until analysis.
The samples were analyzed by gas chromatography (GC) method following an analytical procedure provided by the Sponsor and adapted at Harlan Laboratories Ltd. The test item was used as the analytical standard. Analyzed samples were not discarded without written consent from the study director.
Duplicates were taken of all samples and were stored at Harlan Laboratories Ltd., Itingen, Switzerland. The samples were not discarded without written consent from the study director.

Details on mating procedure:

During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed. The females were removed and housed individually if:
- the daily vaginal smear was sperm positive, or
- a copulation plug was observed.
The day on which a positive mating was determined (copulation plug or sperm) was designated day 0 post coitum. If a female did not mate during the 14-day pairing period, a second pairing of this female with a male in the same group, which had already mated successfully, was considered. All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.

Duration of treatment / exposure:

Males: Minimum 4 weeks
Females: Approximately 7 weeks

Frequency of treatment:

Daily at approximately 24 hour intervals.

Duration of test:

After a minimum of 43 days treatment (on Day 47 of the study) for males; on day 5 post partum for females.

No. of animals per sex per dose:

12/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a 28-day toxicity study in Han Wistar rats, (Harlan Laboratories UK study no. 0930-0125), using dose levels of 0, 15, 150 and 1000 mg/kg bw/day, resulting in a NOEL (no-observed-effect-level) of 1000 mg/kg bw/day.
- Rationale for animal assignment (if not random): Randomisation was performed after at least three days of acclimatization using a computer-generated random algorithm. Body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality was recorded twice daily. Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded daily from treatment start to day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
Males: Weekly during pre-pairing and after pairing periods.
Females: Pre-Pairing period days 1 - 8 and 8 - 14, gestation period days 0 - 7, 7 - 14 and 14 - 21 post coitum and lactation period days 1 - 4 post partum.
No food consumption was recorded during the pairing period.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day: Males were sacrificed when they were no longer needed for the assessment of reproductive effects (on day 47 of the study). Dams and pups were sacrificed on day 5 post partum. If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.
- Organs examined: At the scheduled sacrifice, organs were weighted and preserved. Organs were trimmed from any adherent tissue, and their wet weight taken. If not indicated otherwise, organs were preserved in neutral phosphate buffered 4 % formaldehyde solution.

The following tissues were collected and examined:
Epididymides (fixed in modified Davidson's solution)*
Ovaries
Uterus (incl. oviducts, cervix and vagina)
Mammary gland
Prostate gland and seminal vesicles incl. coagulating glands
Testes (fixed in modified Davidson's solution)*
Pituitary
All gross lesions

The following tissues were also weighed:
Epididymides (fixed in modified Davidson's solution)*
Testes (fixed in modified Davidson's solution)*
Pituitary
* Testes and epididymides were weighed separately.

At the scheduled sacrifice, all animals were weighed and sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated. All parent animals, except those excessively cannibalized, were examined macroscopically for any structural changes at the scheduled necropsy. For the parent animals, special attention was directed at the organs of the reproductive system. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4 % formaldehyde solution.

OTHER:
Histotechnique
All organ and tissue samples to be examined by the principal investigator were processed, embedded and cut at an approximate thickness of 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis was stained with PAS. Special stains were used at the discretion of the study pathologist.

Histopathology
Slides of all organs and tissues collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the principal investigator. The same applied to all occurring gross lesions.
Qualitative assessment of the male reproductive organs was performed. Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.
Histological examination of all organs was carried out on the organs listed above for any females that did not give birth and all infertile males of all dose groups.
A histopathology peer review was performed.

Ovaries and uterine content:

The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

Fetal examinations:

All pups, except those excessively cannibalized, were examined macroscopically for any structural changes at the scheduled necropsy.
The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post partum.

Statistics:

For reproduction data, group mean values were calculated both on a litter basis and on a percentage per group basis. Mean pup weights were calculated from the individual weights both on a per group and on a per litter basis.

Means and standard deviations of various data were calculated and included in the
report.
The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
All animals survived until the scheduled necropsy. No clinical signs were observed during the study.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No effect on food consumption was noted in males in any dose group. Food consumption of females was not affected by the treatment with the test item.
Body weight of males was not affected by the treatment with the test item. At 1000 mg/kg/day, the body weight gains were statistically significantly increased for a few days during pre-pairing and after pairing periods and the difference in body weights were statistically significantly increased between days 1 and 8 in the pre-pairing period but these were considered not to be test item-related.

Pre-Pairing, Pairing, Gestation and Early Lactation Periods:
No test item-related effects on body weights and body weight gains of females were observed at any dose level.
The body weights of females were statistically significantly increased on days 2 and 5 of the lactation period.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Mating Performance and Fertility
No effect on mating performance or fertility was observed at any dose level.
With exception for one female in the control group (no. 58) mating of all females was recorded during the first pairing period. The pre-coital time was not affected by the treatment with the test item. Mean (median) pre-coital times calculated for the first pairing period were 3.3 (3), 2.7 (3), 3.0 (3) and 2.7 (3) days in order of ascending dose levels. One female in the control group (no. 50), three in group 2 (nos. 61, 63, 66), four females (nos. 73, 76, 77, 79) in group 3 and two females (nos. 86, 88) in group 4 were not pregnant. Fertility indexes (number of females achieving pregnancy as a percentage of females paired) and conception rates (number of females achieving pregnancy as a percentage of females mated) were 91.7 %, 75.0 %, 66.7 % and 83.3 in the control, group 2, group 3 and group 4, respectively.

All pregnant females gave birth to living pups. Gestation index (number of females with living pups as a percentage of females pregnant) was 100% in all dose groups.

Duration of Gestation
No effects on gestation length were observed at any dose level. Mean duration of gestation was 21.6, 21.4, 21.6 and 21.8 days, in order of ascending dose level.

Corpora Lutea Count
No effects on corpora lutea count were observed at any dose level. Mean number of corpora lutea per dam was 14.8, 13.9, 14.0 and 14.1 in order of ascending dose levels.

Implantation Rate and Post-Implantation Loss
No effects on implantations or post-implantation loss were observed. The number of implantations was slightly lower in group 4 but it was not statistically significant and the mean value (11.9) was within the historical background data (10.5 – 14.4).

Litter Size at First Litter Check
No effects on litter size were noted in any dose group at first litter check. The overall mean numbers of living pups per dam at first litter check were 12.7, 11.9, 12.3 and 11.2, whereas birth indexes (number of pups borne alive as a percentage of implantations) were 92.1 %, 92.2 %, 95.1 % and 94.1 % at the dose levels of 0, 30, 300 and 1000 mg/kg bw/day, respectively.

Postnatal Loss Days 0 - 4 Post Partum
No effect on post natal loss was noted between days 0 and 4 post partum. Two pups from group 1 (litter no. 51) were missing and one pup from group 2 (litter no. 70) had spontaneous death on day 2 post partum.


ORGAN WEIGHTS (PARENTAL ANIMALS)
No test item-related finding on organ weights of males or females were noted.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No macroscopical findings were observed at necropsy.

HISTOPATHOLOGY (PARENTAL ANIMALS)

OTHER FINDINGS (PARENTAL ANIMALS)
There were no treatment-related findings.
In particular, qualitative examination of the stages of spermatogenesis in the testis did not reveal any treatment-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle. No treatment-related microscopic abnormalities were observed in the evaluation of the ovarian follicles and corpora lutea of the ovaries.

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

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
CLINICAL SIGNS (OFFSPRING)
No test item-related observations were noted in pups during the first litter check or during the early lactation at any dose level. In the control group one pup had wound on the neck region from day 2 to day 4 post partum. No observations of pups were noted in the test item-treated groups.

BODY WEIGHT (OFFSPRING)
Body Weights to Day 4 Post Partum:
No effects on pup body weights were noted at any dose level. Mean body weights of pups on day 1 post partum were: 6.2 g, 6.3 g, 6.5 g and 6.8 g, at the dose levels of 0, 30, 300 and 1000 mg/kg/day, respectively, body weight gain of pups during the first four days of the lactation period was +44.5%, +47.5%, +49.3% and +52.8%, respectively.

GROSS PATHOLOGY (OFFSPRING)
No test item-related findings were noted at macroscopic examination of F1 pups. For one male pup in control and for one female pup in group 3 the sore skin was recorded.

OTHER FINDINGS (OFFSPRING)
Pups sex ratio was not affected by exposure to the test item at any dose level. At first litter check, percentages of male pups were 51 %, 51 %, 40 % and 50 %, in order of ascending dose level.

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: developmental toxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

P Animals Breeding for F1 Litters

Group (mg/kg/day)	1 (0)	2 (30)	3 (300)	4 (1000)
Female numbers	49 – 60	61 – 72	73 – 84	85 – 96
No. of females paired	12	12	12	12
Number of females mated	11	12	12	12
Number of pregnant females (A)	11	9	8	10
Number of non-pregnant females (B)	1	3	4	2
Number of females, which lost their litters	0	0	0	0
Numbers of females, which did not deliver any pups	0	0	0	0
Number of females which reared their pups until day 5post partum	11	9	8	10

(A) Female No. 58 did not mate with male no. 9 in the first pairing period but mated with male no. 10 in the second pairing period.

(B) Female no. 50 in group 1, females 61, 63, 66 in group 2, female nos. 73, 76, 77, 79 in group 3 and female nos. 86, 88 in group 4 were not pregnant.

 

Conclusions:

No test item-related effects were observed on body weights, body weight gains, food consumption of males and females at the dose level up to and including the dose 1000 mg/kg bw/day. At 1000 mg/kg/day, the body weight gains were statistically significantly increased for a few days during pre-pairing and after pairing periods and the difference in body weights were statistically significantly increased between days 1 and 8 in the pre-pairing period but these were considered of not toxicological relevance.
No effects on mating performance, fertility, corpora lutea count or duration of gestation were observed at any dose level. No effects on implantations or post-implantation loss were observed.
No test item-related findings were noted at first litter check and during lactation in pups up to day 4 post partum at any dose level.
During necropsy and following histopathological examination changes no test item-related findings were noted.
Based on these results, NOEL (No Observed Effect Level) for general toxicity and for reproduction/ developmental toxicity in males and females was considered to be 1000 mg/kg bw/day, the highest does level tested.

Executive summary:

Introduction

The purpose of this study was to generate preliminary information concerning the effects of Spiroglycol (SPG) on male and female reproductive performance such as gonadal function, mating behavior, conception and parturition.

Four groups of 12 males and 12 females were treated by gavage with Spiroglycol (SPG) once daily. Males were treated over a 14-day pre-pairing period and during the pairing period up to one day before necropsy. Females were treated throughout the pre-pairing, pairing, gestation and lactation period up to the day 4post partum.

The following dose levels were used:

Group 1: 0 mg/kg body weight/day (control group)

Group 2 30 mg/kg body weight/day

Group 3: 300 mg/kg body weight/day

Group 4 1000 mg/kg body weight/day

A standard dose volume of 5 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (Peanut Oil).

 

Parent Animals

Mortality and General Tolerability

All animals survived the scheduled study period. No test item-related clinical signs were noted at any dose level.

Food Consumption

Food consumption was not affected by the treatment with the test item at any dose level.

Body Weights

Body weights and body weight gains of males and females were not affected by the treatment up to and including the dose level of 1000 mg/kg body weight/day.

Reproduction and Breeding Data

No effects on mating performance, fertility, corpora lutea count, duration of gestation, post-implantation loss, litter size or breeding loss were observed at any dose level.

Organ Weights

No effects on measured organ weights were noted in males and females.

Macroscopic / Microscopic Findings

There were no treatment-related findings.

 

Litter Data - F1 Pups

Findings at First Litter Check and during Lactation

No test item-related findings were noted in pups at any dose level. Pups sex ratio was not affected by the exposure to the test item at any dose level.

Pup Weights to Day 4Post Partum

No effects on pup body weights or body weight gain were noted at any dose level.

Macroscopic Findings

At necropsy of pups, there were no abnormal findings.

Microscopic Findings

There were no treatment-related findings.

Conclusion

Based on these results, NOEL (No Observed Effect Level) for general toxicity and for reproduction/ developmental toxicity in males and females was considered to be 1000 mg/kg bw/day, the highest does level tested.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

The purpose of this study was to generate preliminary information concerning the effects of the test substance on male and female reproductive performance such as gonadal function, mating behaviour, conception and parturition. The test item was administered once daily orally (by gavage) to male and female rats throughout the pre-pairing and pairing periods, after pairing (males), gestation and early lactation periods (females) including the day before scheduled necropsy.

 

The study is a valid investigation of the toxicological effects resulting from repeated oral gavage administration of the test item to rats. The test substance was administered in peanut oil as vehicle at dosages of 30, 300, and 1000 mg/kg body weight/day, and controls received the vehicle only. The test substance was administered to male rats for 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4post partum.

 

No test item-related findings were noted at first litter check and during lactation in pups up to day 4post partumat any dose level.

 

During necropsy and following histopathological examination changes no test item-related findings were noted.

Based on these results, NOEL (No Observed Effect Level) for general toxicity and for reproduction/ developmental toxicity in males and females was considered to be 1000 mg/kg bw/day, the highest does level tested.

Justification for selection of Effect on developmental toxicity: via oral route:
A reproduction/developmental toxicity screening test was performed to assess the effects of the test substance on male and female reproductive performance such as gonadal function, mating behaviour, conception and parturition. The test was performed to GLP according to OECD Guideline 421 in an appropriate test species.

Justification for classification or non-classification

Reproductive toxicity includes adverse effects on sexual function and fertility in adult males and females as well as developmental toxicity in the offspring. Reproductive toxicity is subdivided under two main headings, namely adverse effects on sexual function and fertility and adverse effects on development of the offspring.

Adverse effects on sexual function and fertility

Adverse effects on sexual function and fertility includes any effect of substances that has the potential to interfere with sexual function and fertility. This includes, but is not limited to alterations to the female and male reproductive system, adverse effects on onset of puberty, gamete production and transport, reproductive cycle normality, sexual behaviour, fertility, parturition, pregnancy outcomes, premature reproductive senescence or modifications in other functions that are dependent on the integrity of the reproductive systems.

 

Adverse effects on development of the offspring.

Developmental toxicity includes any effect which interferes with the normal development of the conceptus, either before or after birth and resulting from either parent prior to conception or exposure of the offspring during prenatal development, or post-natally to the time of sexual maturation. However, it is considered that classification under the heading of developmental toxicity is primarily intended to provide a hazard warning for pregnant women and for men and women of reproductive capacity. Therefore, for pragmatic purposes of classification, developmental toxicity essentially means adverse effects induced during pregnancy or as a result of parental exposure. These effects can be manifested at any point in the life span of the organism. The majority of manifestations of developmental toxicity include death of the developing organisms, structural abnormality, altered growth and functional deficiency.

The reproduction/developmental toxicity screening test indicated no test item-related effects were observed on body weights, body weight gains, food consumption of males and females at the dose level up to and including the dose 1000 mg/kg bw/day. Furthermore, no effects on mating performance, fertility,corpora luteacount or duration of gestation were observed at any dose level. No effects on implantations or post-implantation loss were observed. No test item-related findings were noted at first litter check and during lactation in pups up to Day 4post partumat any dose level.Finally, necropsy and following histopathological examination, no test item-related findings were noted.