Cyclohexanemethanol, 4-(1-methylethyl)-

Toxicity to reproduction

Administrative data

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:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

[(1r,4r)-4-(propan-2-yl)cyclohexyl]methanol; [(1s,4s)-4-(propan-2-yl)cyclohexyl]methanol

EC Number:

939-719-8

Cas Number:

5502-75-0

Molecular formula:

C10H20O

IUPAC Name:

[(1r,4r)-4-(propan-2-yl)cyclohexyl]methanol; [(1s,4s)-4-(propan-2-yl)cyclohexyl]methanol

Test material form:

other: Colourless liquid

Details on test material:

- Name of test material (as cited in study report): Mayol [Chemical name: Reaction Mass of Cis-4-(isopropyl) cyclohexanemethanol
and Trans-4-(isopropyl) cyclohexanemethanol]
- Physical state: Colourless liquid
- Analytical purity: 99.8% (sum of 2 isomers)
- Lot/batch No.: 1001098671
- Storage condition of test material: At ambient temperature, protected from light.

Test animals

Species:

rat

Strain:

other: Crl:CD (SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd
- Age at study initiation: 70 days old (F0 animals)
- Weight at study initiation: Males: 317 g to 378 g; Females: 232 g to 267 g
- Housing: Solid (polycarbonate) bottom cages were used acclimatisation, gestation, littering, lactation and maturation periods. Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing. The number of animals per cage was as follows:
(a) Pre-pairing: up to 5 animals;
(b) During pairing: 1 male and 1 female;
(c) Males after pairing: up to 5 males;
(d) Gestation: 1 female; and
(e) Lactation: 1 female + litter
- Diet (e.g. ad libitum): Ad libitum. SDS VRF1 Certified pelleted diet. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water (e.g. ad libitum): Ad libitum. Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
- Acclimation period: Five days before treatment commenced.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23 ºC
- Humidity (%): 40-70%
- Photoperiod (hrs dark / hrs light): 12 hours light : 12 hours dark.

IN-LIFE DATES: From: 3 December 2012 (beginning of treatment) To: 21 January 2013 (necropsy of F0 males) or 14 January 2013 to 19 January 2013 (necropsy of F0 females)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Starting with the lowest concentration, approximately 50% of the final volume of vehicle was added to the required amount of the substance and magnetically stirred until all of the test material had dissolved. The remaining amount of vehicle was added to make up the required total volume. The formulation was then returned to the container and mixed using a high shear homogeniser to produce a homogeneous suspension. Remaining concentrations were formulated in ascending order using the same method.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: Up to 2 weeks
- Proof of pregnancy: Ejected copulation plugs. Sperm within vaginal smear (wet smear using pipette lavage).
- After successful mating each pregnant female was caged (how): Individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and stability of the test material in the vehicle was demonstrated over a period of up to 15 days of refrigerated storage (2-8°C); and for 24 hours of ambient storage. Samples of each formulation prepared for administration on Day 1 and during the final week of treatment were analysed for achieved concentration of the test substance.

Duration of treatment / exposure:

The substance, was administered as follows:
F0 Males: Minimum of 5 weeks, including 15 days before pairing.
F0 Females: 15 days before pairing until Day 6 after birth of F1 generation (animals of the F1 generation were not dosed).

Frequency of treatment:

Once/day

Details on study schedule:

Male and female F0 animals received the test article from 15 days before pairing until termination. The F0 females were allowed to litter and rear their offspring and were killed on Day 7 of lactation. F0 males were killed following successful littering by the females. A similarly constituted control group received the vehicle, corn oil, at the same volume-dose. The F1 generation received no direct administration of the test substance; any exposure was in utero or via the milk.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

10 Males and 10 females receiving 0 (control), 50, or 150 mg/kg/day, and 12 males and 12 females receiving 300 mg/kg/day.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of a 14-day rat preliminary toxicity study (Huntingdon Life Sciences study number: HIK0015) the high dose was set at 300 mg/kg/day. In that study there was essentially an absence of motile or progressively motile sperm among all males receiving the highest dose of 500 mg/kg/day and one out of five males receiving 300 mg/kg/day. Sperm motility was apparently unaffected at 50 or 150 mg/kg/day. The high dose was set at 300mg/kg/day based on the observations of toxic effects, per OECD guideline 421, and the intermediate and low doses were set at intervals to allow determination of any dose related trends.

Positive control:

Not required.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Performed on each animal to monitor general health at the following time points: (a) All adults: Once each week; and (b) F0 Females: Days 0, 7, 14 and 20 after mating and Days 1 and 7 of lactation.

BODY WEIGHT: Yes
- Time schedule for examinations: (a) F0 animals: During acclimatisation, the day that treatment commenced (Week 0), weekly thereafter and on the day of necropsy; and (b) F0 females after mating: Days 0, 3, 7, 10, 14, 17 and 20 after mating and Days 1, 4 and 7 of lactation.

FOOD CONSUMPTION :
The weight of food supplied, that remaining and an estimate of any spilled was recorded as follows: (a) F0 animals: Weekly until paired for mating; and (b) F0 females after mating: Days 0-2, 3-6, 7-9, 10-13, 14-16 and 17-19 after mating and Days 1-3 and 4-6 of lactation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

Oestrous cyclicity (parental animals):

Dry smears: Vaginal smears were taken daily for 15 days before pairing, using cotton swabs moistened with saline. The smears were subsequently examined to establish the duration and regularity of the oestrous cycle.

Wet smears: After pairing with the male, daily smearing was continued using pipette lavage, until evidence of mating was observed.

Sperm parameters (parental animals):

Immediately after scheduled sacrifice of each male, the left vas deferens, epididymis and testis was removed and the epididymis and testis were weighed. The following tests were performed:
(a) Sperm motility A sample of sperm was expressed from the vas deferens; the percentages of motile and progressively motile sperm and sperm motion parameters were reported;
(b) Sperm morphology: An aliquot of the sperm was examined for the assessment of sperm morphology. The percentages of normal, decapitate and abnormal sperm were reported;
(c) Sperm count: The left cauda epididymis of each male was weighed and assessed for sperm count. The concentration (million/g) and total number of sperm were reported. The concentration (million/g) and total number of sperm were reported; and
(d) Homogenisation-resistant spermatids count: The concentration (million/g) and total number of sperm were reported.

Litter observations:

Parturition observations: From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.

Records made during littering phase include the following:
(a) Clinical observations: Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate;
(b) Litter size: Daily records were maintained of mortality and consequent changes in litter size on Days 1-7 of age;
(c) Sex ratio: The sex ratio of each litter was recorded on Days 1, 4 and 7 of age; and
(d) Individual offspring bodyweights: Recorded on Days 1, 4 and 7 of age.

Postmortem examinations (parental animals):

All F0 adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

Time of necropsy was as follows:
(a) F0 males: After successful littering by females; and
(b) F0 females: (1) Scheduled kill - Day 7 of lactation; (2) Failing to mate - Day 25 after last day of pairing; (3) Failing to produce viable litter - Day 25 after mating; and (4) Litter death before Day 7 of lactation - on or after day last offspring dies.

For F0 females, the following were recorded:
(a) Each uterine horn: Number of implantation sites;
(b) Females failing to produce a viable litter: The number of uterine implantation sites were checked; and
(c) Female whose litter died before Day 7 of lactation: Mammary tissue appearance.

Requisite organs (i.e., epididymides, prostate, seminal vesicles, and testes in males and ovaries in females) were weighed for adult animals killed at scheduled necropsy.

Histopathology examinations were carried out for the testes, epididymides, ovaries, and any abnormal tissues.

Postmortem examinations (offspring):

F1 offspring scheduled kill at Day 7 of age.

The F1 animals were:
(a) Examined externally: Those offspring deemed normal were discarded without further macroscopic examination. Any externally abnormal offspring were examined internally and any abnormal tissues were retained; and
(b) Premature deaths (before Day 7 of age): Missing offspring and those grossly autolysed or grossly cannibalised could not be examined. All other offspring dying before Day 7 of age were examined as detailed above except that the cranial roof was not removed unless required to investigate a cranial abnormality; this also included an assessment for the presence of milk in the stomach, where this was possible.

Statistics:

Significant differences between Control and treated groups were expressed at the 5% (p<0.05) or 1% (p<0.01) level. Statistical methods used include analysis of variance (followed by pairwise t-tests), Cochran-Armitage test, Linear-by-linear association test, Shirley’s test, and Wald’s test.

Reproductive indices:

Oestrous cycles: The percentage of F0 females showing the following classifications of oestrous cycles before pairing were recorded:
(a) Regular: All observed cycles of 4 or 5 days;
(b) Irregular: At least one cycle of 2, 3 or 6 to 10 days; and
(c) Acyclic: At least 10 days without oestrus.

Pre-coital intervals were tabulated for females only, for the time elapsing between initial pairing and mating. Percentage of females with pre-coital intervals was calculated for durations of 1-4, 5-8, 9-12 and 13-14 days of pairing.

Mating performance and fertility: Individual data was tabulated and group values were calculated for males and females separately for the following:
(a) Percentage mating;
(b) Conception rate (%); and
(c) Fertility index (%).

Gestation length: Calculated as the number of gestation days up to and including the day on which offspring were first observed, with Day 1 = day of mating for calculation purposes. Where parturition had started overnight, this value was adjusted by subtracting half of one day.

Offspring viability indices:

Litter size: Group mean litter sizes were calculated from the individual litter values.

Survival indices: The following were calculated for each litter:
(a) Post-implantation survival index (%);
(b) Live birth index (%); and
(c) Viability index (%).

(Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded. Additionally, group mean values were calculated from individual litter values.)

Sex ratio: The percentage of male offspring in each litter was calculated at Day 1, and for live offspring on Days 1 and 7 of age.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female receiving 150 mg/kg/day was found dead on Day 2 of lactation. This female exhibited piloerection, underactivity and a hunched posture prior to death. Macroscopic examination did not reveal any abnormal findings. The factor contributing to the death of this animal was undetermined. In the absence of similar signs or deaths during lactation among females receiving a higher dose (300 mg/kg/day), these findings were considered of uncertain relationship to treatment with the substance. One female receiving 300 mg/kg/day was killed for welfare reasons on Day 20 of gestation prior to dose administration. This female had in-life signs consisting of piloerection, underactive behaviour and pallor on Day 20 of gestation. Macroscopic examination revealed an oedematous pancreas, pale areas on the liver and abnormal/reduced caecal contents. The correlating microscopic findings were slight oedema and moderate degranulation of acinar cells of the pancreas. The factor contributing to the death of this animal was undetermined. In the absence of similar signs or macropathology findings among the other females in this group, a relationship to treatment is uncertain.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Group mean bodyweight for females receiving 300 mg/kg/day was significantly lower (6.7% lower) on Day 20 of gestation when compared with Control group. Correspondingly, overall group mean bodyweight gain for females receiving 300 mg/kg/day during Days 0-20 of gestation was lower when compared with Control values (49.3% gain vs. 55.4% gain in Controls); this was mainly due to the lower weight gain between Days 17 and 20 of gestation. Group mean bodyweight for females receiving 300 mg/kg/day was significantly lower than Control group on Days 4 and 7 of lactation (8.4% and 7.2% lower than Control, respectively). Group mean bodyweight gain during Days 1-7 of lactation was lower for females receiving 150 or 300 mg/kg/day (35% and 32% lower, respectively) when compared with Controls, without statistical significance. Group mean food intake was marginally decreased for females receiving 300 mg/kg/day between gestation Day 7-9 and 17-19 of gestation. Group mean food consumption during Days 1-3 of lactation for females receiving 150 or 300 mg/kg/day was statistically significantly lower than that of the Controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Group mean bodyweight for females receiving 300 mg/kg/day was significantly lower (6.7% lower) on Day 20 of gestation when compared with Control group. Correspondingly, overall group mean bodyweight gain for females receiving 300 mg/kg/day during Days 0-20 of gestation was lower when compared with Control values (49.3% gain vs. 55.4% gain in Controls); this was mainly due to the lower weight gain between Days 17 and 20 of gestation. Group mean bodyweight for females receiving 300 mg/kg/day was significantly lower than Control group on Days 4 and 7 of lactation (8.4% and 7.2% lower than Control, respectively). Group mean bodyweight gain during Days 1-7 of lactation was lower for females receiving 150 or 300 mg/kg/day (35% and 32% lower, respectively) when compared with Controls, without statistical significance. Group mean food intake was marginally decreased for females receiving 300 mg/kg/day between gestation Day 7-9 and 17-19 of gestation. Group mean food consumption during Days 1-3 of lactation for females receiving 150 or 300 mg/kg/day was statistically significantly lower than that of the Controls.

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:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No findings in the tissues examined (male and female reproductive organs: testes, epididymides, ovaries, and abnormal tissues).

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Oestrus cycles were considered unaffected by treatment with the substance.

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

Treatments at 300 mg/kg/day resulted in marked reductions in sperm motility, the majority of sperm motion parameters and the percentage of normal sperm with associated increases in percentage of static and decapitate sperm. Slight reductions in cauda epididymal sperm numbers were also evident. These changes were statistically significant. No such effects were observed at the 50 or 150 mg/kg/day dose levels. There was no effect on sperm counts within the testis at any dose level.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Pre-coital interval was considered unaffected by the substance. Conception rate and fertility index were statistically significantly lower for animals receiving 300 mg/kg/day, when compared with Controls; percentage mating was unaffected.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Parental toxicity NOAEL is based on lower body weight and food consumption in females and sperm effects in males.

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 300 mg/kg/day, offspring in 3 litters were observed to have little or no milk in stomach on Day 1 post partum and subsequently several pups in each of these litters died, and in 2 of these litters the pups were also recorded as being cold to touch.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

The offspring viability index was significantly lower among females receiving 300 mg/kg/day when compared with Controls. The lower offspring viability index reflected decreases in litter size up to Day 7 of age; namely, litter size was reduced among two females receiving 300 mg/kg/day. Litter size was reduced to a lesser extent (loss of one or two pups) at 150 mg/kg/day compared to Controls, however, statistical significance was only attained on Day 4 of age at 150 mg/kg/day.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Group mean bodyweight on Days 1, 4 and 7 of age and mean bodyweight gain were decreased in male (statistically significant in some instances) and female offspring from dams treated at 300 mg/kg/day when compared with Controls. The body weights of surviving male and female offspring from one litter were lower than expected on Day 1 of age. At 50 and 150 mg/kg/day there were marginal reductions in bodyweight (5.5% to 8.2% less than Control on Day 7 of age) and bodyweight gain (7.8% to 14.3% less than Control over Days 1-7 of age) of male and female offspring. In one litter, which had the most pup deaths, body weights of surviving male and female offspring were lower than expected on Day 1 of age.

Food consumption and compound intake (if feeding study):

not examined

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

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reproductive and developmental toxicity. NOAEL based on consideration of effects on pups during the first 7 days of lactation.

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Gestation

Gestation length was within the expected time frame of 22 to 23 days for all females receiving 50 or 150 mg/kg/day, and the majority of females receiving 300 mg/kg/day. One female receiving 300 mg/kg/day had a gestation length of 21.5 days which is unusual.

Gestation length and gestation index were considered unaffected by treatment for females receiving 50, 150, or 300 mg/kg/day. There was a slight decrease in gestation index (7 live births from 8 females, or 88% when compared to the Control group and this was not statistically significant), excluding one female, the death of which was considered to be undetermined/incidental and hence data from this female was not included in the assessment of the gestation index.

Littering

Taking into account that one female receiving 150 mg/kg/day was found dead on Day 2 of lactation, one female receiving 300 mg/kg/day was killed for welfare reasons on Day 20 of gestation, three females receiving 300 mg/kg/day were not pregnant, one female had total litter resorption, and one female had total litter loss on Day 1 of lactation, the data on littering is based on the 10, 10, 9 and 6 litters for evaluation in the Control group, and at 50, 150 and 300 mg/kg/day the substance, respectively.

Implantation counts were reduced in females receiving the substance at 300 mg/kg/day when compared with Controls. 

Group mean post implantation survival index was considered to be unaffected by treatment at any dose.

Live birth index was considered to be unaffected by treatment with the substance at 300 mg/kg/day among litters with offspring surviving to Day 7 of age, when compared with Controls, although it was slightly lower than expected at 150 mg/kg/day (92.2%, although not statistically significant), reflecting an increased number of litters with losses of pups, especially in one litter. 

Applicant's summary and conclusion

Conclusions:

In the OECD 421, the NOAEL was considered by the study director to be 50 mg/kg bw/day since at 150 mg/kg/day the live birth index was slightly lower than in Controls, and there was one litter with several pup deaths where offspring bodyweights on Day 1 of age were lower than expected, and this was also seen at 300 mg/kg/day. However, the reduction in the live birth index seen at 150 mg/kg bw/day is not statistically significant and there was no similar reduction at 300 mg/kg bw/day, therefore the effects are not considered to be toxicologically relevant. The hypothesis mentioned in the report for the litter which had the most pup deaths, was a possible adverse effect on this litter in utero. However, in the OECD 414 study there were no effects on pup development up to 300 mg/kg bw/day. The OECD 414 results confirm that the effects seen at 150 mg/kg bw/day in the OECD 421 study are not related to in utero exposure, and are not a direct toxic effect of the substance. Based on this, the NOAEL (developmental toxicity) for the OECD 421 can be set to 150 mg/kg bw/day. The reductions in post natal survival and growth of the offspring at 300 mg/kg bw/day are most likely associated with the reduced maternal food consumption recorded at the start of lactation, and/or the presence of the substance in the milk affecting pup suckling behaviour (unpleasant taste to the milk), which may explain why a number of pups in several litters at 300 mg/kg/day were observed with no milk in the stomach. A reasonable speculation could be made that there could well be an effect of the fragrance ingredient on the maternal behaviour of the dams towards the pup.

Executive summary:

Study Synopsis

The potential for reproductive toxicity due to the substance was evaluated in a GLP-compliant screening assay conducted to the OECD Testing Guideline No. 421 (Huntingdon Life Sciences, 2013). The substance was administered orally by gavage at dose levels of 50, 150 or 300 mg/kg/day to groups of 10 male and female Crl:CD (SD) rats for a period of at least seven weeks (12 males and 12 females at the 300 mg/kg/day dose level), including assessment of reproductive and developmental effects.

There were no post-dose or clinical signs considered to be related to dosing with the substance. Mean bodyweight and bodyweight gain were similar to the concurrent Control for males after 7 weeks of treatment.

Effects on bodyweight and body weight gain were as follows: overall group mean bodyweight gain was lower when compared with Controls, for females receiving 300 mg/kg/day during Days 0-20 of gestation. Group mean bodyweight for females receiving 300 mg/kg/day was significantly lower than the Control group on Days 4 and 7 of lactation (8.4% and 7.2% lower than Control, respectively). Group mean bodyweight gain during Days 1-7 of lactation was lower, but not statistically significant, for females receiving 150 or 300 mg/kg/day (35% and 32% lower, respectively) when compared with Controls. Mean food consumption was marginally decreased for females receiving 300 mg/kg/day during Days 7-9 and 17-19 of gestation, and statistically significantly lower during Days 1-3 of lactation for females receiving 150 or 300 mg/kg/day when compared with Controls.

One female receiving 150 mg/kg/day (3F 64) was found dead on Day 2 of lactation and one female receiving 300 mg/kg/day (4F 82) was killed for welfare reasons on Day 20 of gestation. The factor contributing to the deaths of these animals was undetermined, and in isolation of any other deaths at these dose levels the relationship to treatment is uncertain.

There were no effects of the substance on oestrous cycles and pre-coital intervals, and no effect on the ability of animals to mate.

Treatment at 300 mg/kg/day markedly impaired the reproductive function of the animals: no pregnancy resulted from the mating of three pairs and the litter from a fourth pair died in utero. At the end of dosing, there were no treatment-related macroscopic or microscopic abnormalities detected in the right testis, right epididymis or ovaries. However, as measured at the end of the study, treatments at 300 mg/kg/day resulted in a marked reduction in sperm motility, sperm motion parameters and the percentage of normal sperm. Slight reductions in cauda epididymal sperm numbers were also evident. The aetiology of the sperm findings is unclear as there were no associated histopathological findings and no correlating organ weight effects.

The mean number of implantations at 300 mg/kg/day was markedly lower than in Controls (and this contributed to decreased litter size) but there was no effect of treatment on the post implantation survival or live birth index among litters surviving to Day 7 of age. One female receiving 300 mg/kg/day had a shorter than expected gestation length of 21.5 days compared to the expected gestation length of 22 to 23 days.

As would be expected the pups in this litter had low bodyweights on Day 1 of age. All pups in this litter were observed to have no milk in the stomach on Day 1 of age and five pups subsequently died. Lower than expected pup bodyweights at Day 1 of age were also recorded in two other litters in the 300 mg/kg/day group. Overall, the offspring viability index was decreased, reflecting the death of 4 or more pups in 2 litters between Days 1 and 7 of age, and litter size on Day 7 of age was markedly lower than in Controls. Decreased group mean offspring bodyweights and weight gains were observed between Days 1 and 7 of age compared to Control groups. The decreased litter sizes as observed on Day 1 of age were considered to be responsible for the lower maternal body weight gain during gestation, particularly between Day 17 and Day 20 of gestation when the foetuses would have been growing rapidly. These reductions in post natal survival and growth of the offspring may be associated with the reduced maternal food consumption recorded at the start of lactation, and/or the presence of the substance in the milk affecting pup suckling behaviour, which may explain why a number of pups in several litters at 300 mg/kg/day were observed with no milk in the stomach. It should be noted that the effect of the odour of the substance on the behaviour of the dams towards the pups is unknown.

At 150 mg/kg/day, the live birth index was slightly decreased compared to Controls, reflecting the death of 12 offspring in 4 litters compared with the death of a single pup in the Controls. While the live birth index represented the largest losses of pups (i.e., on Day 1), litter size up to Day 7 of age also was slightly decreased (only statistically significant on Day 4). In one litter which had the most pup deaths, bodyweights of surviving male and female offspring were lower than expected on Day 1 of age suggesting a possible adverse affect on this litter in utero and this was similar to the situation in a litter of the 300 mg/kg/day group. As a result, a relationship to treatment could not be ruled out. Group mean offspring bodyweights and weight gains also were marginally decreased between Days 1 and 7 of age compared to Controls. These reductions in post natal survival, Day 1 pup bodyweights in some litters, and growth of the offspring may be associated with the reduced maternal food consumption recorded at the start of lactation, and/or the presence of the substance in the milk affecting pup suckling behaviour. As noted above, the effect of the odour of the substance on the behaviour of the dams towards the pups is unknown.

At 50 mg/kg/day, there was no adverse effect on the survival, growth or development of the offspring.

Based on the results, it was concluded that the No-Observed-Adverse-Effect Level (NOAEL) for systemic toxicity in male and female Crl:CD(SD) rats is 150 mg/kg/day administered by oral gavage, and the oral (gavage) NOAEL for reproductive and developmental toxicity is 50 mg/kg/day, since at 150 mg/kg/day the live birth index was slightly lower than in Controls, and there was one litter with several pup deaths where offspring bodyweights on Day 1 of age were lower than expected and this was also seen at 300 mg/kg/day.  

Discussion of Sperm-related Effects

Regarding the effects on sperm motility and male fertility, it is important to note that sperm motility findings are well known to result from the production of oxidative stress via reactive oxygen species (ROS) at the testes. The testes are considered highly sensitive to oxidative stress and vulnerable to effects of oxygen depletion due to the nature of the activities occurring in this tissue (spermatogenesis) and a limited oxygen supply (Bansal and Bilsapuri, 2011; Aitken and Roman, 2008). A link between oxidative stress and sperm or testicular effects has been identified to occur under certain conditions (e.g., hypobaric hypoxia) and exposure to certain compounds (Farias et al., 2012; Morakinyo et al., 2011; Shin et al., 2010; Aitken and Roman, 2008). Among the well-known compounds that induce both oxidative stress and sperm findings is ethanol, is a primary alcohol (Aitken and Roman, 2008; Tremellen, 2008). Indeed, ethanol is a well-known testicular toxin (Maneesh et al., 2006; Wu and Cederbaum, 2004), creating oxidative stress at the testes (Schlorff et al., 1999; Nordmann et al., 1992). 

In a situation that is likely to be analogous to the events that occur with ethanol, it is considered that the substance, a primary alcohol, may also cause oxidative stress at the testes. The substance is a small organic molecule (156.27 g/mol) with a cyclohexyl ring and a methyl group attached at the para-position. The alcohol is the sole functional group and attached to the methyl group (see IUCLID section 1). Like ethanol, the substance is a primary alcohol and under normal conditions of exposure (inhalational, dermal) is likely to undergo metabolism in the liver in the same manner as ethanol (a simple alcohol) and perillyl alcohol (a terpenoid primary alcohol). The metabolism of the alcohol in the liver yields oxidation products of the alcohol group, an oxidized ring compound, and glucuronidated metabolites, the majority of which would be readily excreted in the urine (JECFA, 2003, 2000). This oxidation requires oxygen being delivered from the blood. Predictive modelling of the metabolism of the substance has indicated that this may indeed occur in vivo (see IUCLID Section 7.1). Briefly, in silico modelling under in vitro and in vivo conditions has indicated that three metabolites are likely to be produced in the liver via tertiary C-aliphatic oxidation to produce a tertiary/primary diol and C-aliphatic oxidation of the original primary alcohol to produce an aldehyde and then a carboxylic acid. The rapid oxidative metabolism may induce oxidative stress, including at the testes, which in turn may cause depletion of local glutathione (GSH) levels. GSH is a key molecule in the protection of sperm from damage that may be caused by the presence of ROS.

With respect to the oral route of administration, the substance is without any ionisable groups and has physico-chemical characteristics that would indicate a rapid and high level of absorption from the gastrointestinal (GI) tract, supported by the available repeat dose toxicity studies as well as the developmental and reproductive toxicity studies for the registered substance. The water solubility is estimated to be 214 mg/L and the log Pow (octanol:water coefficient) is 3.45, which is optimal for diffusion across lipid membranes; both of these parameters together with the small size of the molecule favour absorption of the substance from the GI tract (ECHA, 2012; Martinez and Amidon, 2002). Under conditions leading to high peak plasma levels, such as would occur with high oral gavage bolus doses of the substance, the usual hepatic metabolic pathways would be overloaded, resulting in high levels of the substance and possibly its metabolites in the systemic circulation and subsequently lead to extra-hepatic metabolism of the compound. In particular, testicular metabolism of alcohol in rats has been shown to result in the production of aldehydes and free radicals (Quintans et al., 2005). In addition to the production of free radicals/ROS, the high oxygen demands of metabolism will cause oxygen depletion and lipid peroxidation at the testes (Quintans et al., 2005; Rosenblum et al., 1989). The recent work of Morakinyo et al. (2011) also supports the hypothesis that oxidative stress at the testes can produce adverse effects on sperm. In this study, adverse effects on sperm motility and male fertility caused by oral gavage doses of the calcium channel blockers nifedipine, verapamil, or diltiazem were associated with the production of oxidative stress at the testes. The fertility and sperm findings occurred in the absence of any histopathological findings in the testes, epididymal tubule, or seminal vesicle and were fully reversed after a 30-day non-dosed (recovery) period. Based on these results, and given the lack of any noted morphological or tissue damage in the testes following dosing in rats, the sperm motility effects of the substance are expected to be both temporary and reversible. In support of reversibility of the sperm effects, it is noted that such effects of cyclohexanol in rabbits were fully reversible (Dixit et al., 1980). Another recent study established an association between the sperm motility effects of diethyl maleate and the production of testicular oxidative stress in mice dosed intraperitoneally, daily for two weeks (Kaur et al., 2006). Altogether, based on the above-mentioned arguments, it is accepted that the oxidative stress produced at the testes would lead to effects on the sperm and testes.  

Taking into consideration the gavage dosing, the sperm motility and associated fertility effects observed in the reproductive toxicity screening study are considered to be due to oxidative stress occurring as a result of testicular metabolism in the absence of sufficient oxygen for glucuronidation, because of high plasma levels of the substance and its metabolites following oral gavage administration of a high bolus dose. The substance Terpineol (EC No.: 232-268-1) provides an example of this phenomenon, as oral gavage doses of 750 mg/kg bw resulted in sperm-related effects, but these effects were not seen when the same dose levels were administered via the diet (ECHA, 2013). The hypothesis that the sperm effects seen with the substance are secondary effects due to the route of administration is further supported by the fact that as confirmed with microscopic (histopathologic) evaluations, the substance did not display intrinsic toxicity in the form of tissue damage to male (or female) reproductive organs and tissues at the highest dose in this study. Indeed, the registrant has concluded that the substance is not classified for reproductive toxicity or effects on or via lactation.

Discussion of Offspring-related Effects

Regarding the decreases in offspring body weights at 150 and 300 mg/kg/day compared to Controls on Day 1, the slight decrease in live birth rate at 150 mg/kg/day, and decreased litter sizes at both 150 and 300 mg/kg/day during lactation, it is noted that there were decreased body weight gains and food consumption in females of the 150 and 300 mg/kg/day dose groups compared to Controls, including on the first day of the lactation period. Therefore it seems likely that these events are related, although it is unclear whether the lower number of pups per litter contributed to the decreased maternal body weight gain or vice versa. Importantly, as noted by the Study Director, the effect of the substance on the palatability of the breast milk and subsequently on suckling behaviour of the pups is unclear, as is the effect of this odiferous compound on the behaviour and interrelationship of the dams and pups, given the reliance on olfactory cues between dams and pups.  Therefore, it is difficult to be certain that there is a direct toxic effect of the substance on offspring. 

Overall Conclusion

Based on the results of this reproductive and developmental toxicity screening study, the NOAEL for systemic toxicity in male and female rats is 150 mg/kg/day administered by oral gavage, given the observations of effects on sperm motility at the higher dose level of 300 mg/kg/day. The oral (gavage) NOAEL for reproductive and developmental toxicity is 50 mg/kg/day, based on observed effects on the post natal survival of pups and reductions in Day 1 pup body weights in the higher dose groups. However, the effects on sperm are not clearly related to a direct effect of the substance on testicular tissues or sperm, but may be secondary to the effects of oxidative stress at the kidneys, given the oral gavage bolus dose route used in the study. Moreover, the effect of the odour of the test substance on the behaviour of the dams, the interactions between dams and pups, and the suckling activities of the pups is unknown and, as such, there is uncertainty as to a direct toxicity to the pups.

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