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REACH

Cyclohexanemethanol, 4-(1-methylethyl)-

EC number: 939-719-8 | CAS number: 5502-75-0

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

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.

Two additional studies have been conducted for the substance:

- An oral gavage screening study was conducted to assess the potential for effects on the testes in rats. The study showed systemic toxicity and mortalities at the high dose of 1,000 mg/kg bw/day, including oligospermia. Minimal effects on sperm were observed at the low dose of 50 mg/kg bw/day.

-An exploratory study was conducted to assess the potential for effects in male rats following 14 days of administration via the dietary route. The study was extended to 18 days in duration and showed a decrease in sperm effects compared to the oral gavage route of administration used in other studies. All of the observed effects on sperm were shown to be reversible.

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:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

GLP compliance:

yes

Limit test:

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)

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.

Dose / conc.:

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

Dose / conc.:

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

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.

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.

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):

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: 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.

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.

Critical effects observed:

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

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Generation:

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.

Critical effects observed:

Reproductive effects observed:

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.

Conclusions:

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.

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.

References

-Aitken RJ, Roman SD. Antioxidant systems and oxidative stress in the testes. 2008. Oxid Med Cell Longev 1:15-24.

-Bansal AK, Bilaspur GS. Impacts of oxidative stress and antioxidants on semen functions. 2011. Vet Med Inter 2011:1-7.

-Dixit VP, Gupta RS, Kumar S, Joshi BC. Reversible chemical sterilization: effects of cyclohexanol administration on the testes and epididymides of male rabbit. 1980. In J Physiol Pharmac 24(4): 278-286.

-ECHA. Guidance on information requirements and chemical safety assessment. Chapter R.7c: Endpoint specific guidance. November 2012 (version 1.1). Guidance for implementation of REACH. European Chemicals Agency.

- ECHA. Terpineol Registration Dossier. 2013. Available at: http://apps.echa.europa.eu/registered/data/dossiers/DISS-9d944137-bb01-5d50-e044-00144f67d249/DISS-9d944137-bb01-5d50-e044-00144f67d249_DISS-9d944137-bb01-5d50-e044-00144f67d249.html.

-Farias, JG, Puebla M, Acevedo A, Tapia PJ, et al. Oxidative stress in rat testis and epididymis under intermittent hypobaric hypoxia: protective role of ascorbate supplementation. J Androl 2010;31(3):314-321.

-JECFA. Evaluation of Certain Food Additives. Fifty-first report of the Joint FAO/WHO Expert Committee on Food Additives. WHO Technical Report Series 891. 2000. Available at:http://whqlibdoc.who.int/trs/WHO_TRS_891.pdf.

-JECFA. Alicyclic primary alcohols, aldehydes, acids, and related esters. Safety evaluation of certain food additives / prepared by the fifty-ninth meeting of the Joint FAO/WHO Expert Committee on Food Additives. WHO Food Additive Series: 50. 2003. Available at:http://www.inchem.org/documents/jecfa/jecmono/v50je10.htm.

-Kaur P, Kalia S, Bansal MP. Effect of diethyl maleate induced oxidative stress on male reproductive activity in mice: Redox active enzymes and transcription factors expression. 2006. Molec Cell Biochem 291:55-61.

-Martinez MN, Amidon GL. A mechanistic approach to understanding the factors affecting drug absorption: a review of fundamentals. J Clin Pharmacol 2002;42:620-643.

-Maneesh M, Dutta S, Chakrabarti A, et al. Alcohol abuse-duration dependent decrease in plasma testosterone and antioxidants in males. 1999. In J Physiol Pharmacol 50:291-296. Cited in: Aitken and Roman, 2008.

-Morakinyo AO, Iranloye BO, Daramola AO, Adegoke OA. Antifertility effect of calcium channel blockers on male rats: association with oxidative stress. 2011. Adv Med Sci 56(1):95-105.

-Nordmann R, Ribiere C, Rouach H. Ethanol-induced lipid peroxidation and oxidative stress in extrahepatic tissues. 1990. Alcohol Alcohol 25:231-237. Cited in: Aitken and Roman, 2008.

-Quintans LN, Castro GD, Castro JA. Oxidation of ethanol to acetaldehyde and free radicals by rat testicular microsomes. Arch Toxicol 2005;79:25-30.

-Rosenblum ER, Gavaler JS, Van Thiel DH. Lipid Peroxidation: a mechanism for alcohol-induced testicular injury. Free Radic Biol Med 1989;7(5):569-577.

-Schlorff EC, Husain K, Somani SM. Dose and time dependent effects of ethanol on anti-oxidant system in rat testes. 1999. Alcohol. 18:203-214. Cited in: Aitken and Roman, 2008.

-Shin I-S, Lim, J-H, Kim, S-H, Kim K-H,et al. Induction of oxidative stress in the epididymis of rats after subchronic exposure to epichlorohydrin. Bull Environ Contam Toxicol 2010;84:667-671.

-Tremellen K. Oxidative stress and male fertility – a clinical perspective. 2008. Hum Reprod Update 14(3):243-258.

-Wu D, Cederbaum AI. Alcohol, Oxidative Stress, and Free Radical Damage. 2004. National Institute on Alcohol Abuse and Alcoholism. Available at:http://pubs.niaaa.nih.gov/publications/arh27-4/277-284.htm.

Effect on fertility: via oral route

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEL
: 150 mg/kg bw/day
Study duration:: subacute
Species:: rat
Quality of whole database:: The information is reliable and consistent with the database as a whole.

Effect on fertility: via inhalation route

Endpoint conclusion:: no study available

Effect on fertility: via dermal route

Endpoint conclusion:: no study available

Additional information

Summary of Data

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, 2013a). 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 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. 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 only on the results from this study, 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.

It should be noted that dose levels for the reproductive toxicity screening study were selected based on a 14-day oral gavage toxicity study in the same strain of rats (Huntingdon Life Sciences 2013b). In the 14-day study, the daily gavage administration of the substance at doses up to and including 500 mg/kg/day was well tolerated. However, computer-assisted sperm analysis (CASA) revealed treatment-related effects on sperm motility, sperm concentration, and total number of epididymal spermatids at dose levels of 300 and/or 500 mg/kg/day. Testicular sperm numbers were unaffected at all dose levels. Although it was considered likely that a high dose level of 500 mg/kg/day would be tolerated for the duration of the reproductive toxicity screening study, in view of the sperm effects detected in the preliminary oral gavage study, and taking into consideration the longer period of dosing in the reproductive toxicity screening study, the high dose level for the reproductive toxicity screening study was set below 500 mg/kg/day (i.e., 300 mg/kg/day).

In an additional exploratory study in rats using the dietary route of administration (Huntingdon Life Sciences, 2014), male rats dosed daily at dose levels of 0 (control), 1500, 3000, 5000, and 7500 ppm (equivalent to approximately 0, 109, 214, 353, and 493 mg/kg/day) for a total of 18 days also showed sperm effects related to vigour and motion, as also seen in the oral gavage studies. However, it was seen that the magnitude of the effects observed at the two higher doses (~353 and 493 mg/kg/day in the male rats) was slightly less than observed in the previous studies using oral gavage dosing at approximately equivalent or lower dose levels (300 and 500 mg/kg/day). In all studies irrespective of route of administration, there were no microscopic correlates with the changes in sperm motility (i.e., there was no evidence of effects on reproductive organ tissues). Moreover, data from recovery group animals in the dietary study indicate that the effects on the sperm were reversible and, given sufficient time post-dose, complete recovery might have been observed in previously-dosed animals. Based on the results of this investigative dietary study, it was concluded that the treatment-related effects observed on the sperm of male rats exposed to the substance by oral gavage dosing could be slightly reduced by employing dietary administration and that the lower magnitude of sperm effects following dietary as compared to after oral gavage dosing would suggest that future preclinical studies with the substance could be conducted using dietary administration.

In general, the findings of sperm-related effects of the substance were reflected in a limited male reproductive toxicity screening study that was conducted previously at only two dose levels (BASF Experimental Toxicology and Ecology, 2010). In the 2010 study, there were no significant effects at 50 mg/kg/day, consistent with the results of the full reproductive screening study (Huntingdon Life Sciences, 2013a). Sperm-related toxicities such as oligospermia were observed at 1000 mg/kg/day (a dose level more than three times greater than that evaluated in the OECD 421 screening study), along with testicular tubular degeneration. However, the contribution of the severe levels of systemic toxicity of the substance (resulting in early morbidities and mortalities) at the high dose to testicular tubular degeneration is unclear, and no further details were available from the study report.

Discussion of Effects on Male Fertility

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. Consistent with evidence of hepatic metabolism of the oral gavage bolus doses, the 28-day repeated dose oral gavage study in rats showed liver effects indicative of a hepatic response of adaptation change (vacuolation in the absence of any degenerative liver changes). 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 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. Furthermore, as shown in the examinations of male rats in the 14-day dietary study, administration of the substance via the diet results in a decrease in the incidence and severity of sperm effects. The 14-day dietary study also re-confirmed that the substance does not cause morphological damage to the organs, including the reproductive organ tissues, or the spermatogenic process, and showed that the sperm effects on vigour and motility are reversed upon cessation of treatment.

This hypothesis is supported by the recently performed dietary OECD TG 408 study (Envigo, 2017), in which no adverse effects were observed up to 3000 ppm (eq. to 188 mg/kg bw/day for males and 220 mg/kg bw for females). In the shorter-term OECD TG 407 and 421 reproductive toxicity screening study, both via gavage dose, effects were observed on sperm motility at similar or higher levels. These effects were considered to be specifically associated with bolus dosing. To confirm that much higher plasma levels occur after oral gavage dosing than after dietary dosing, plasma levels of Mayol were measured during the OECD TG 408 dietary study and the oral gavage OECD TG 414 study. Though the full report is not yet available the evidence indicates that via gavage the peak plasma levels of Mayol are much higher than in the dietary study. These high plasma levels of Mayol show that the oral absorption is fast and high. It is anticipated that at levels < 1 mMol (ca 200 mg/kg bw) Mayol is metabolised/oxidised into its acid and conjugated. At higher levels Mayol as such escapes first pass metabolism in the liver and enters the systemic circulation and direct exposure to the male reproductive system occurs. All organs have the capability to further oxidise substances and also these organs. Compared to other organs the testes is somewhat hypoxic. When significant amounts of substance is oxidised within the testes the oxygen levels may be depleted (oxidative stress) such that it can affect sperm viability. As these effects are only seen at high dose levels that exceed a threshold that cannot be envisaged after exposure to humans, and metabolic overload is anticipated, these effects are not considered as relevant reproductive hazard effects.

Discussion of Effects on Female Fertility

Based on the available information, there are no data clearly indicating effects on female fertility. There were no effects on oestrous cycles. The observed effect on decreased numbers of pregnancies and litter sizes is consistent with the effects on sperm motility in males, which is considered to have been affected by oxidative stress due to testicular metabolism of the substance following oral gavage administration of a high bolus dose. At the high dose level, there were no effects on post implantation survival or live birth index among surviving litters. As a result, the NOAEL for female fertility could be considered to be 300 mg/kg/day.

It is worth noting that possible effects on offspring body weights on Day 1 at the high and mid-doses, on the slight decrease in live birth rate at the mid-dose, and on litter sizes during lactation to Day 7 may have been due to decreased body weight gains and food consumption in the maternal animals, including on the first day of the lactation period. Importantly, these pup effects may have been due to a possible effect of the substance on the palatability of the breast milk and subsequently on suckling behaviour of the pups or an 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. In support of the possibility that the substance may have been widely distributed (resulting in its presence in breast milk, or in the pores of the skin), the findings from a 28-day general toxicity oral gavage study in rats indicated the possibility of adaptive changes in the liver, which in turn may have signaled an overloading of hepatic metabolism. As a result, both the test substance and/or its metabolites may have entered into the circulation to become widely distributed. If this occurs with the test substance, then the available data would indicate that oral gavage dosing at levels greater than 100 mg/kg/day may lead to widespread distribution of the substance and/or its metabolites.

Overall Conclusion for Effects on Fertility

Overall, there is uncertainty as to an intrinsic effect of the substance on fertility in male animals, as the effects on sperm motility at the high dose are consistent with oxidative stress occurring as a result of testicular metabolism of the substance in the absence of sufficient oxygen for glucuronidation. Such metabolism in the absence of sufficient oxygen may be due to high plasma levels of the substance and its metabolites following oral gavage administration of a high bolus dose. There is no clear evidence of effects on female fertility, taking into consideration a lack of effects on oestrus cycles at all dose levels and that findings such as decreased litter sizes and reduced numbers of pregnancies at the high dose level are consistent with the effects observed on sperm motility in the males. Consequently, while the Study Director concluded that the NOAEL for systemic toxicity in male and female rats is 150 mg/kg/day administered by oral gavage, it is considered that, based on all available data and reasonable arguments regarding uncertainty as to a direct toxicity to the animals, the NOAEL for reproductive and developmental effects based on the reproduction/developmental toxicity screening study can be considered to be 300 mg/kg/day for parental animals.

Further studies are being conducted to explore the metabolism and toxicology of the substance.

References

-Aitken RJ, Roman SD. Antioxidant systems and oxidative stress in the testes. 2008. Oxid Med Cell Longev 1:15-24.

-Bansal AK, Bilaspur GS. Impacts of oxidative stress and antioxidants on semen functions. 2011. Vet Med Inter 2011:1-7

-BASF Experimental Toxicology and Ecology. Summary Report. Screening study on testes toxicity in male Wistar rats. Project No. 06R0725/06056. 04 March 2010.

-ECHA. Terpineol Registration Dossier. Accessed at:http://apps.echa.europa.eu/registered/data/dossiers/DISS-9d944137-bb01-5d50-e044-00144f67d249/DISS-9d944137-bb01-5d50-e044-00144f67d249_DISS-9d944137-bb01-5d50-e044-00144f67d249.html.

-Farias, JG, Puebla M, Acevedo A, Tapia PJ, et al. Oxidative stress in rat testis and epididymis under intermittent hypobaric hypoxia: protective role of ascorbate supplementation. J Androl 2010;31(3):314-321.

- Huntingdon Life Sciences, Mayol: Reproductive/Developmental Toxicity Screening Study in the CD Rat by Oral Gavage Administration. Project No. HIK0019. 27 November 2013a.

- Huntingdon Life Sciences, Mayol: Preliminary Toxicity Study by Oral Gavage Administration to Crl:CD(SD) Rats for 14 Days. Project No. HIK0015. 10 April 2013b.

- Huntingdon Life Sciences, 14-Day Investigative Toxicity Study by Dietary Administration to Crl:CD(CD) Rats with a 4-Week Recovery Period. Project No. HIK0023. 20 February 2014.

-JECFA. Alicyclic primary alcohols, aldehydes, acids, and related esters. Safety evaluation of certain food additives / prepared by the fifty-ninth meeting of the Joint FAO/WHO Expert Committee on Food Additives. WHO Food Additive Series: 50. 2003. Available at: http://www.inchem.org/documents/jecfa/jecmono/v50je10.htm.

-Maneesh M, Dutta S, Chakrabarti A, et al. Alcohol abuse-duration dependent decrease in plasma testosterone and antioxidants in males. 1999. In J Physiol Pharmacol 50:291-296. Cited in: Aitken and Roman, 2008.

-Martinez MN, Amidon GL. A mechanistic approach to understanding the factors affecting drug absorption: a review of fundamentals. J Clin Pharmacol 2002;42:620-643.

-Morakinyo AO, Iranloye BO, Daramola AO, Adegoke OA. Antifertility effect of calcium channel blockers on male rats: association with oxidative stress. 2011. Adv Med Sci 56(1):95-105.

-Nordmann R, Ribiere C, Rouach H. Ethanol-induced lipid peroxidation and oxidative stress in extrahepatic tissues. 1990. Alcohol and Alcoholism 25:231-237. Cited in: Aitken and Roman, 2008.

-Quintans LN, Castro GD, Castro JA. Oxidation of ethanol to acetaldehyde and free radicals by rat testicular microsomes. Arch Toxicol 2005;79:25-30.

-Rosenblum ER, Gavaler JS, Van Thiel DH. Lipid Peroxidation: a mechanism for alcohol-induced testicular injury. Free Radic Biol Med 1989;7(5):569-577.

-Schlorff EC, Husain K, Somani SM. Dose and time dependent effects of ethanol on anti-oxidant system in rat testes. 1999. Alcohol. 18:203-214. Cited in: Aitken and Roman, 2008.

-Shin I-S, Lim, J-H, Kim, S-H, Kim K-H, et al. Induction of oxidative stress in the epididymis of rats after subchronic exposure to epichlorohydrin. Bull Environ Contam Toxicol 2010;84:667-671.

-Tremellen K. Oxidative stress and male fertility – a clinical perspective. 2008. Hum Reprod Update 14(3):243-258.

-Wu D, Cederbaum AI. Alcohol, Oxidative Stress, and Free Radical Damage. 2004. National Institute on Alcohol Abuse and Alcoholism. Available at: http://pubs.niaaa.nih.gov/publications/arh27-4/277-284.htm.

Effects on developmental toxicity

Description of key information

A prenatal developmental toxicity study was performed according to OECD Guideline 414 and in compliance with GLP, three groups of twenty female rats received test substance, formulated in corn oil, at doses of 30, 100 or 300 mg/kg bw/day by the oral gavage route of administration at a dose volume of 4 mL/kg bw/day, from Day 6 to Day 19 after mating, inclusive. A similarly constituted Control group received the vehicle, at the same volume dose over the same period. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination. During the study blood samples were taken from selected animals in each group on Days 6 and 19 after mating and were used for toxicokinetic evaluation. Clinical observations, post-dosing observations, body weight and food consumption measurements were performed. Adult females were examined macroscopically at necropsy on Day 20 after mating and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 414 without any deviation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (inspected on 01-03 December 2015/ signed on 15 February 2016)

Limit test:

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 1002451619
- Physical state: Colourless liquid
- Expiration date of the lot/batch: 1 January 2017
- Purity test date: 24 November 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At ambient temperature, protected from light

Species:

rat

Strain:

other: Crl:CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: Approximately 71 days old
- Weight at study initiation: 220-295g
- Housing: Acclimatization - up to four animals; During pairing - one (stock) male and one female; Gestation - one female; Solid (polycarbonate) bottom cages were used during the acclimatization and gestation periods. Grid bottomed polypropylene cages were used during pairing.
- Diet: SDS VRF1 Certified pelleted diet. ad libitum
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum
- Acclimation period: 5 days before commencement of pairing

DETAILS OF FOOD AND WATER QUALITY:
No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 40-70 %
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated; minimum of 15 air changes per hour
- Photoperiod: 12 hours light : 12 hours dark

IN-LIFE DATES: From: 8 June 2016 To:

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Method of preparation: Starting with the lowest concentration, the required amount of test item was placed into a suitable container to which 50% of the final volume of vehicle was added. This was magnetically stirred until all of the test item was uniformly mixed and then made up to the required volume. This was then placed on a high shear homogeniser until homogenous.
- Frequency of preparation: Weekly, and may be prepared in advance of the first day of dosing.
- Storage of formulation: Refrigerated (nominally 2-8 °C).
- The homogeneity and stability of formulations during storage was determined as part of another study, Huntingdon Life Sciences Report No. HIK0016. These investigations demonstrated that formulations in the concentration range of 1 and 250 mg/mL were homogenous and stable for 15 days following refrigerated storage (2-8 ˚C) and for 24 hours following ambient storage.

VEHICLE
- Concentration in vehicle: 7.5, 25 and 75 mg/L
- Amount of vehicle (if gavage): 4 mL/kg bw/day

DOSE VOLUME: 4 mL/kg bw/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity: Homogeneity and stability of the test material in the vehicle in the concentration range of 1 and 250 mg/mL had been demonstrated over a period of up to 15 days following refrigerated storage (2-8 ˚C) and for 24 hours following ambient storage in Huntingdon Life Sciences Report No. HIK0016.
Achieved concentration: Samples of each formulation prepared for administration on Day 6 (Phase 1) and Day 19 (Phase 2) after mating were analyzed for achieved concentration of the test item.

Results: The mean concentrations of test substance in test formulations analyzed during the study were within 4% of the nominal concentration, confirming the accuracy of formulation preparation. Differences from mean values were within 1% confirming precise analysis. Procedural recoveries were within 1 % of nominal confirming continued accuracy of the analytical method.

Details on mating procedure:

- Impregnation procedure: co-housed
- If co-housed:
- M/F ratio per cage: 1:1 with identified stock males
- Proof of pregnancy: Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm referred to as Day 0 of pregnancy
A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals; males were approximately 25-30 weeks at the time of mating.

Duration of treatment / exposure:

Females: Day 6 to 19 after mating, inclusive

Frequency of treatment:

Once daily at approximately the same time each day.

Duration of test:

Animals were killed on Day 20 after mating.

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

20 females/dose
Phase 1: 6 females/dose
Phase 2: 14 females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels used in this study, to investigate for effects on embryo-fetal development (OECD414) (0, 30, 100 and 300 mg/kg bw/day), were selected in conjunction with the Sponsor based on the results of a 14-day preliminary study (Huntingdon Life Sciences Study No. HIK0015), a 28-day repeat dose toxicity study (Huntingdon Life Sciences Study No. HIK0016), and a reproductive/developmental toxicity screening study (OECD 421; Huntingdon Life Sciences Study No. HIK0019), all of which were performed via oral gavage administration.
In the 14-day study, dose levels of 50, 150, 300, 500, 750 and 1000 mg/kg bw/day were investigated. The doses of 750 and 1000 mg/kg bw/day were found to exceed the maximum tolerated dose, with a marked decline in clinical condition observed after three or four doses, requiring premature sacrifice of the animals, and these doses were considered unsuitable for further investigation. The dose levels up to and including 500 mg/kg bw/day were well tolerated for the full 14-day period with no unscheduled deaths. Treatment related effects were limited to a suggestion of slightly low weight gain in females receiving 500 mg/kg bw/day and slightly increased liver weight in females at 300 or 500 mg/kg bw/day.
In the 28-day study treatment with the test item at 30, 100 or 300 mg/kg bw/day was well tolerated in females with adverse effects in the liver noted at 300 mg/kg bw/day.
In the OECD 421 study, dose levels of 50, 150 and 300 mg/kg bw/day were well tolerated during the 2-week pre-pairing period and during gestation. Test item-related effects during these periods were limited to a reduction in mean body weight gain during Days 17-20 of gestation for females given 300 mg/kg bw/day. One female given 300 mg/kg bw/day was killed for reasons of animal welfare on Day 20 of gestation following a marked decline in clinical condition, however this premature death was considered to be of uncertain relationship to treatment in view of the isolated incidence of the clinical signs and macroscopic abnormalities detected for this animal, and no factors contributing to death were determined following histopathological evaluation of the retained tissues.
Based on the information available, a dose level of 300 mg/kg/day was considered appropriate to use as the high dose level in Phase 1 of this embryo-fetal development study; higher dose levels were anticipated to cause excessive reduction in weight gain of the females and concomitant confounding effects. Dose levels of 30 and 100 mg/kg/day were selected as the low and intermediate doses to establish a suitable dose response relationship for any treatment-related changes and to give an approximate 3-fold interval between doses.
As no adverse effects of treatment were observed at 300 mg/kg bw/day in Phase 1, an additional 14 females per group were used to form Phase 2 of the study in order to form the full complement of 20 females at each dose level.

- Rationale for animal assignment: On the day of positive evidence of mating (Day 0). Only females showing at least two copulation plugs were allocated.

Maternal 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 animal ill-health amongst the occupant(s). During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed observations were recorded daily at the following times in relation to dose administration: Pre-dose; 1 to 2 hours after completion of dosing; As late as possible in the working day.
A detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each adult was recorded on Days 0, 3, 6 and then daily until termination.

FOOD CONSUMPTION: Yes
- The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 inclusive after mating.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on Day 20 after mating; Animals were killed by Carbon dioxide asphyxiation with subsequent exsanguination.
- Necropsy: All 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.

OTHERS:
Toxicokinetics: Blood samples (nominally 0.5 mL) were collected from animals on Day 6 and Day 19 after mating at the different time points (1, 2, 4, 8 and 24 hours after dosing).

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes; Gravid uterine weight (including cervix and ovaries)
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number of live/dead fetuses: Yes

Fetal examinations:

SACRIFICE:
Method of kill for fetuses: Chilling on a cool plate (approximately 0 °C)

FETAL EXAMINATION AND PROCESSING
Examination of all viable fetuses and placentae: Dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Examined externally with abnormalities recorded. The sex of each fetus was recorded.
Examination of nominally 50% of fetuses in each litter: Sexed internally and eviscerated.
Fixation: Subjected to a gross internal examination were fixed in Industrial Methylated Spirit (IMS).
Remaining fetuses were fixed whole in Bouin’s fluid.
Processing: Bouin’s fixed fetuses were subject to free-hand serial sectioning.
IMS fixed fetuses were processed and stained with Alizarin Red.

FETAL PATHOLOGY EXAMINATION
Bouin’s fixed fetuses: Serial sections were examined for visceral abnormalities.
Alizarin Red stained fetuses: Assessed for skeletal development and abnormalities.

Statistics:

See "Any other information on materials and methods incl. tables"

Indices:

Pre-implantation loss (%) = [(Number of corpora lutea – Number of implantations) / Number of corpora lutea] x 100
Post-implantation loss (%) = [(Number of implantations – Number of live fetuses) / Number of implantations] x 100

Historical control data:

See "Attached background material" section

Clinical signs:

no effects observed

Description (incidence and severity):

The administration of test substance at dose levels up to and including 300 mg/kg bw/day was well tolerated and there were no unscheduled deaths.
There were no test item-related clinical signs apparent at routine physical examination or signs in association with dose administration among females at any dose level investigated.

Mortality:

no mortality observed

Description (incidence):

No mortality was observed.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- During the 24-hour period after the first dose administration, between Day 6 and Day 7 of gestation, females given 300 mg/kg bw/day showed minor but statistically significant mean body weight loss of 2 grams compared to mean weight gain of 2 grams in Controls. Thereafter, mean body weight gain for these females was essentially similar to Control, such that overall mean weight gain from Day 6-20 of gestation was 98% of Control. Mean body weight gain of females given 30 or 100 mg/kg bw/day was unaffected by treatment.
- There was no effect of test substance administration on mean gravid uterine weight or net mean body weight gain at any dose level investigated.
- Body weight change Days 6-20: 109 ± 202, 115 ± 13.4, 111 ± 15.5 and 107 ± 12.3 at 0, 30, 100 and 300 mg/kg bw/day.
- Adjusted body weight change Days 6-20: 26 ± 7.3, 26 ± 10.3, 24 ± 10.4 and 22 ± 8.7 at 0, 30, 100 and 300 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Test substance administration at 300 mg/kg bw/day was associated with slightly low food intake when compared to Controls during Days 6-9 of gestation [19 g/animals/day vs 21 g/animals/day in control group], with statistical significance attained; thereafter, mean food intake for these females was essentially similar to Controls. The transient and slightly reduced food consumption and body weight gain were considered to have likely been caused by the taste of the test item. There was no effect of test substance administration on mean food consumption at 30 or 100 mg/kg bw/day. Full data information are provided in the attached tables of the study report.

Food efficiency:

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

Description (incidence and severity):

There was no effect of test substance administration on mean gravid uterine weight at any dose level investigated.
- Gravid uterine weight: 84 ± 20.8, 89 ± 10.9, 87 ± 10.4 and 85 ± 9.7 at 0, 30, 100 and 300 mg/kg bw/day.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test substance-related macroscopic abnormalities detected at scheduled termination on Day 20 of gestation.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Number of abortions:

no effects observed

Description (incidence and severity):

- Corpora lutea (mean): 17.5 ± 2.65, 17.0 ± 2.50, 17.2 ± 2.48 and 17.6 ± 2.37 (/20) at 0, 30, 100 and 300 mg/kg bw/day.
- Implantations (mean): 15.4 ± 2.44, 16.3 ± 2.10; 15 ± 1.85 and 16.2 ± 1.66 (/20) at 0, 30, 100 and 300 mg/kg bw/day.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

- Pre-implantation loss (mean): 2.1, 0.7, 1.6 and 1.4 (/20 females) at 0, 30, 100 and 300 mg/kg bw/day.
- Pre-implantation loss (%): 11.2, 4.7, 8.5 and 8.4 at 0, 30, 100 and 300 mg/kg bw/day.
- Post-implantation loss (mean): 1.3, 0.8, 0.9 and 1.1 (/20 females) at 0, 30, 100 and 300 mg/kg bw/day.
- Post-implantation loss (%): 9.6, 4.6, 5.3 and 6.9 at 0, 30, 100 and 300 mg/kg bw/day.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

None observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

- Early resorptions (mean): 1.3, 0.8, 0.7 and 1.1 (/20) at 0, 30, 100 and 300 mg/kg bw/day.
- Late resorptions (mean): 0.1, 0.0, 0.2 and 0.0 (/20) at 0, 30, 100 and 300 mg/kg bw/day.
- Total: 1.3, 0.8, 0.9, 1.1 (/20) at 0, 30, 100 and 300 mg/kg bw/day.

Dead fetuses:

no effects observed

Description (incidence and severity):

- Live young males (mean): 7.3 ± 2.31, 7.7 ± 2.62, 8.0 ± 1.93 and 7.0 ± 1.86 at 0, 30, 100 and 300 mg/kg bw/day.
- Live young females (mean): 6.9 ± 2.30, 7.8 ± 2.09, 6.8 ± 2.05 and 8.1 ± 1.61 at 0, 30, 100 and 300 mg/kg bw/day.
- Total Live young (mean): 14.1 ± 3.73, 15.5 ± 2.16, 14.7 ± 1.69 and 15.1 ± 1.96 at 0, 30, 100 and 300 mg/kg bw/day.
- Total Live young (%): 91.6, 95.1, 94.2 and 93.2 at 0, 30, 100 and 300 mg/kg bw/day.

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Not applicable. Animals were killed on Day 20 after mating.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): Not applicable. Animals were killed on Day 20 after mating.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

20/20 females were pregnant at all dose levels.

Other effects:

not specified

Details on maternal toxic effects:

Litter Responses: At scheduled termination on Day 20 after mating, all females were found to be pregnant with live young.
Reproductive Assessment: There was no evidence that maternal treatment with test substance had any adverse effect on litter data, as assessed by the mean numbers of corpora lutea, implantations, resorptions, live young and pre- and post implantation losses, at any dose level investigated.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

early or late resorptions

food consumption and compound intake

gross pathology

maternal abnormalities

mortality

pre and post implantation loss

Remarks on result:

other: No adverse effects were observed.

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

At scheduled termination on Day 20 of gestation, mean fetal weights in the 300 mg/kg/day group were marginally, but statistically significantly, lower than Control (93% of Control for male fetuses, 94% of Control for female fetuses and 93% of Control for overall mean fetal weight); values were below the lower limit of the Historical Control Data (HCD) range (HCD range 3.68-3.92g for males, 3.52-3.77g for females and 3.59-3.84g for overall fetal weight). Mean placental and litter weights at this dose level were essentially similar to Control due to the slightly higher litter size in this group, and it must be noted that the slightly higher litter size (15.1 vs 14.1 in control group) would contribute towards the slightly lower mean fetal weights and as a consequence the magnitude of difference in mean fetal weights were deemed not to be adverse.
There was no effect of maternal treatment with the test substance on mean placental, litter or fetal weights at 30 or 100 mg/kg/day.
- Male fetal weight: 3.78 ± 0.312, 3.76 ± 0.251, 3.75 ± 0.358 and 3.52* ± 0.307 at 0, 30, 100 and 300 mg/kg bw/day.
- Female fetal weight: 3.60 ± 0.271, 3.59 ± 0.227, 3.57 ± 0.324 and 3.38* ± 0.367 at 0, 30, 100 and 300 mg/kg bw/day.
- Overall fetal weight: 3.69 ± 0.286, 3.67 ± 0.239, 3.66 ± 0.328 and 3.44** ± 0.266 at 0, 30, 100 and 300 mg/kg bw/day.

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): At scheduled termination on Day 20 of gestation, mean fetal weights in the 300 mg/kg/day group were marginally, but statistically significantly, lower than Control (93% of Control for male fetuses, 94% of Control for female fetuses and 93% of Control for overall mean fetal weight); values were below the lower limit of the Historical Control Data (HCD) range (HCD range 3.68-3.92g for males, 3.52-3.77g for females and 3.59-3.84g for overall fetal weight). Mean placental and litter weights at this dose level were essentially similar to Control due to the slightly higher litter size in this group, and it must be noted that the slightly higher litter size would contribute towards the slightly lower mean fetal weights and as a consequence the magnitude of difference in mean fetal weights were deemed not to be adverse.
There was no adverse effect of maternal treatment with the test substance on mean placental, litter or fetal weights at 30 or 100 mg/kg/day.
- Male fetal weight: 3.78 ± 0.312, 3.76 ± 0.251, 3.75 ± 0.358 and 3.52* ± 0.307 at 0, 30, 100 and 300 mg/kg bw/day.
- Female fetal weight: 3.60 ± 0.271, 3.59 ± 0.227, 3.57 ± 0.324 and 3.38* ± 0.367 at 0, 30, 100 and 300 mg/kg bw/day.
- Overall fetal weight: 3.69 ± 0.286, 3.67 ± 0.239, 3.66 ± 0.328 and 3.44** ± 0.266 at 0, 30, 100 and 300 mg/kg bw/day.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio, as assessed by the percentage of males per litter, was in line with expectations and unaffected by maternal treatment.
- Sex ratio (%): 51.4, 49.2, 54.2 and 46.1 at 0, 30, 100 and 300 mg/kg bw/day.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

- LItter size (mean): 14.10 ± 3.726, 15.50 ± 2.164, 14.70 ± 1.689 and 15.05 ± 1.959 at 0, 30, 100 and 300 mg/kg bw/day.
- Litter weight (mean): 52.01 ± 14.458, 56.61 ± 6.859, 53.70 ± 6.152 and 51.64 ± 6.729 at 0, 30, 100 and 300 mg/kg bw/day.

Changes in postnatal survival:

not examined

Description (incidence and severity):

Not applicable.

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Description (incidence and severity):

At 300 mg/kg/day there was an increased fetal and litter incidence of the minor abnormalities of delayed/incomplete ossification/ unossified pelvic bones, metacarpals and metatarsals [Pelvic bones: 6, 10, 13 and 41 / Metacarpals: 0, 0, 2, 40 / Metatarsals: 1, 2, 1 and 7 at 0, 30, 100 and 300 mg/kg bw/day], and an increased fetal incidence of delayed/incomplete ossification/ unossified sacrocaudal vertebrae and sternebrae (other than the 5th and/or 6th sternebrae) [Sacrocaudal vertebrae: 8, 12, 15, 30 / Sternebrae other: 3, 11, 7 and 34 at 0, 30, 100 and 300 mg/kg bw/day] when compared to concurrent control and the HCD range. There was also a slightly increased incidence of delayed/incomplete ossification/ unossified cranial centres [4, 4, 13 and 19 at 0, 30, 100 and 300 mg/kg bw/day] when compared to concurrent Controls, however the fetal and litter incidences of this finding were within the HCD range. There were no test item-related fetal findings apparent in litters derived from females given 30 or 100 mg/kg/day.
It was noted that at 30 and 300 mg/kg/day there was an increased incidence of short supernumerary 14th rib compared to concurrent control [4, 14, 4, 20 at 0, 30, 100 and 300 mg/kg bw/day], with the fetal and litter incidence exceeding the HCD range in the 300 mg/kg/day group. In the absence of a clear dose related response, with no similar increased incidence evident in the 100 mg/kg/day group, this finding was considered to be an isolated incidental finding, with no relationship to maternal treatment with the test substance.

Visceral malformations:

no effects observed

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

skeletal malformations

Remarks on result:

other: No adverse effects were observed.

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

The objective of this study was to assess the influence of maternal exposure to the test substance on embryo-fetal survival and development in the Crl:CD(SD) rat when administered by oral gavage at doses of 30, 100 or 300 mg/kg/day from implantation on Day 6 of gestation throughout organogenesis and the fetal growth phases of pregnancy to Day 19 of gestation.

Treatment was well tolerated with no unscheduled deaths. Clinical condition, body weight performance, gravid uterine weight, adjusted body weight gain, food consumption were not adversely affected by the test substance at any dose level investigated, and there were no test item-related macroscopic abnormalities detected at scheduled termination. Test item-related maternal effects were limited to the 300 mg/kg/day group, manifest as minor weight loss following the first dose administration only and slightly low food consumption during Days 6-9 of gestation, however these minor effects were considered not to be adverse at the magnitude observed.

There was no effect of maternal treatment with the test substance on the number of implantations, early or late resorptions, live young, sex ratio or on placental and litter weights at any dose level investigated. At 300 mg/kg/day, mean fetal weights were 6-7% lower than Control although the magnitude of the slight reduction was considered not to be adverse. The lower fetal weight was associated with an increased incidence of delayed/incomplete ossification or unossified pelvic bones, metacarpals, metatarsals, sacrocaudal vertebrae and sternebrae. Changes in the degree of ossification represent a transient stage in fetal development and are therefore considered not to be adverse (Palmer, 1977; Carney and Kimmel, 2007). In this case the differences in mean fetal weight and consequent minor differences in the degree of ossification were considered to be associated with the slightly reduced food consumption and reduced bodyweight gain of the dams during Day 6-9 of gestation.

The transient and slightly reduced food consumption and weight gain were considered to have likely been caused by the taste of the test item.

Based on the results of this embryo-fetal toxicity study, it was concluded that the dose level of 300 mg/kg/day represented the No Observed Adverse Effect Level (NOAEL) for maternal toxicity and for embryo-fetal survival, growth and development.

Conclusions:

Based on the results of this embryo-fetal toxicity study, it was concluded that the dose level of 300 mg/kg bw/day represented the No Observed Adverse Effect Level (NOAEL) for maternal toxicity and for embryo-fetal survival, growth and development.

Executive summary:

In a prenatal developmental toxicity study performed according to OECD Guideline 414 and in compliance with GLP, three groups of twenty female rats received test substance, formulated in corn oil, at doses of 30, 100 or 300 mg/kg bw/day by the oral gavage route of administration at a dose volume of 4 mL/kg bw/day, from Day 6 to Day 19 after mating, inclusive. A similarly constituted Control group received the vehicle, at the same volume dose over the same period. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination. During the study blood samples were taken from selected animals in each group on Days 6 and 19 after mating and were used for toxicokinetic evaluation. Clinical observations, post-dosing observations, body weight and food consumption measurements were performed. Adult females were examined macroscopically at necropsy on Day 20 after mating and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination.

Treatment of female rats with the test substance was well tolerated and there were no unscheduled deaths or test item-related signs observed.

Body weight performance, gravid uterine weight, adjusted body weight gain and food consumption were not adversely affected by the test substance at dose levels up to and including 300 mg/kg/day, and there were no test item-related macroscopic abnormalities detected at scheduled termination. Test item-related maternal effects were limited to the 300 mg/kg/day group, manifest as minor weight loss following the first dose administration only and slightly low food consumption during Days 6-9 of gestation, however effects were minor and considered not to be adverse at the magnitude observed.

There was no effect of maternal treatment with the test substance on litter data, as assessed by the number of implantations, resorptions, live young, sex ratio and pre- and post-implantation losses. Placental and litter weights were similar in all groups. At 300 mg/kg/day, mean male, female and overall fetal weights were 6 and 7% lower than Control respectively and were considered possibly to be related to the slightly reduced food consumption and bodyweight gain of the dams during Days 6-9 of gestation; fetal weights were unaffected by the test substance at 30 or 100 mg/kg/day.

At 300 mg/kg/day there was an increased fetal and litter incidence of the minor abnormalities of delayed/incomplete ossification/ unossified pelvic bones, metacarpals and metatarsals, and an increased fetal incidence of delayed/incomplete ossification/ unossified sacrocaudal vertebrae and sternebrae (other than 5th/6th) when compared to concurrent control and Historical Control Data (HCD). There was also a slightly increased incidence of delayed/incomplete ossification/ unossified cranial centres when compared to concurrent Controls, however the fetal and litter incidences of this finding were within the HCD range and were considered possibly to be associated with the slightly reduced food consumption and bodyweight gain of the dams during Days 6-9 of gestation. There were no test item-related fetal findings apparent in litters derived from females given 30 or 100 mg/kg/day.

Conclusion

Effect on developmental toxicity: via oral route

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEL
: 300 mg/kg bw/day
Species:: rat
Quality of whole database:: Klimisch 1, OECD compliant GLP study.

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

Treatment of female rats with the test substance was well tolerated and there were no unscheduled deaths or test item-related signs observed.

There was no effect of maternal treatment with the test substance on litter data, as assessed by the number of implantations, resorptions, live young, sex ratio and pre- and post-implantation losses. Placental and litter weights were similar in all groups. At 300 mg/kg/day, mean male, female and overall fetal weights were 6 and 7% lower than Control respectively and were considered possibly to be related to the slightly low food consumption of the dams during Days 6-9 of gestation; fetal weights were unaffected by the test substance at 30 or 100 mg/kg/day.

At 300 mg/kg/day there was an increased fetal and litter incidence of the minor abnormalities of delayed/incomplete ossification/ unossified pelvic bones, metacarpals and metatarsals, and an increased fetal incidence of delayed/incomplete ossification/ unossified sacrocaudal vertebrae and sternebrae (other than 5th/6th) when compared to concurrent control and Historical Control Data (HCD). There was also a slightly increased incidence of delayed/incomplete ossification/ unossified cranial centres when compared to concurrent Controls, however the fetal and litter incidences of this finding were within the HCD range and were considered possibly to be associated with the slightly reduced food consumption and bodyweight gain of the dams during Days 6-9 of gestation. The transient and slightly reduced food consumption and body weight gain were considered to have likely been caused by the taste of the test item. There were no test item-related fetal findings apparent in litters derived from females given 30 or 100 mg/kg/day.

Conclusion

Toxicity to reproduction: other studies

Description of key information

In a screening study designed to evaluate potential for effects on male reproductive organs, 5 male rats per group were dosed daily with the substance at 50 or 1,000 mg/kg bw/day. While clinical signs at the low dose were limited to salivation after dosing in 1 of 5 animals on some days, mortality and signs of toxicity were observed at the high dose. Based on histopathological observations of oligospermia in the epididymis and testicular degeneration at 1,000 mg/kg bw/day, the authors determined that the data revealed the potential for adverse effects on male reproductive organs. In the 50 mg/kg bw/day group, significantly reduced total spermatids/g testis (-19%, or 95 +/- 19.3% vs. 117 +/- 13.3% in controls) was observed. While the authors observed a lower % normal sperm (-3%; or 91.1 +/- 3.2% vs. 94.0 +/- 1.3% in controls), this is noted to be a small magnitude of change and unlikely to be biologically significant. There was no statistically significant difference in total sperm/g cauda epididymis, % abnormal sperm, or % motility. It is unclear what role the state of the animals (noting clinical signs and toxicity, including mortality) may have played in the epididymal oligospermia observed at 1,000 mg/kg bw/day. As this was a screening study for toxicity to the testes only, the potential for male reproductive organ toxicity should be further evaluated.

The results of an exploratory 14-day study showed a lower magnitude of effects on the sperm following dietary treatment as compared to oral gavage dosing, which was taken to indicate that toxicology studies with the substance should be conducted using dietary administration. The decrease in magnitude of effects on the adjusted liver weights also indicates that dietary administration should be employed in further toxicology studies.

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:

toxicity to reproduction: other studies

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

12 June 2013 – 22 October 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline available

Principles of method if other than guideline:

Dose range finding oral dietary study with daily dosing to support possible future repeated dose and reproductive toxicity studies using the dietary route.

GLP compliance:

yes

Type of method:

in vivo

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: 57 to 64 days old
- Weight at study initiation: Males: 317 g to 378 g & Females: 224 g to 249 g
- Housing: Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals.
- Diet: Rat and Mouse No. 1 Maintenance Diet. Ad libitum.
- Water: Ad libitum; potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
- Acclimation period: Eight days before commencement of treatment.
- Randomization: randomly allocated.

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

IN-LIFE DATES: From: 7 August 2013 (animal arrival) To: 2 to 3 September 2013 (day of necropsy; main animals) or 30 September 2013 (recovery animals)

Route of administration:

oral: feed

Vehicle:

other: Rat and Mouse No. 1 Maintenance Diet, a powdered diet

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Batches of diet were prepared weekly.

On each occasion of the preparation of the premix, the required amount of test substance was added to an equal amount of diet and stirred. An amount of diet equal to the weight of the mixture was added and the mixture was stirred again until visibly homogenous. This doubling up process was repeated until half the final weight of premix was achieved. This mixture was then ground using a mechanical grinder. The weight of the mixture was then made up to the final weight of the premix with diet. The mixture was then mixed in a Turbula mixer for 100 cycles to ensure the test substance was dispersed in the diet. Aliquots of this premix were then diluted with further quantities of RM1 diet to produce the required dietary concentrations. Each batch of treated diet was mixed for a further 100 cycles in a Turbula mixer.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulation at the highest and lowest concentrations were analysed to assess the stability and homogeneity of the test substance in the diet matrix. The substance stability was demonstrated for 8 days following ambient storage (15-25ºC) and for 22 days following frozen storage (nominally -20ºC) in glass jars. Samples of each formulation prepared for administration in Week 1 of treatment were analysed for achieved concentration of the test substance. Analysis showed that the mean concentrations of the substance in the test formulations were within 3% of the nominal concentrations (within the applied limits of +10%/-15%), which confirmed accurate formulation.

Duration of treatment / exposure:

Continuously for 18 days followed by a 14-day recovery period. While the original study was planned to include 14 days of dosing, there was an initial reduction of food intake during the study up to approximately Day 3 (considered most likely as a consequence of palatability issues), resulting in lower achieved dose levels than expected . From Day 4 onwards the animals showed higher achieved dose levels. As such, the study dosing period was extended to ensure that animals would be exposed to the test substance at the higher dose levels for a full 14 days.

Frequency of treatment:

Continuous

Duration of test:

18 Days

Dose / conc.:

1 500 ppm

Dose / conc.:

3 000 ppm

Dose / conc.:

5 000 ppm

Dose / conc.:

7 500 ppm

No. of animals per sex per dose:

5 Males and 3 females per dose level

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The objective of this study was to assess the systemic toxicity potential of the substance following 14 days of dietary administration in Crl:CD (SD) rats, with particular focus on the male reproductive system, following evidence of toxicity to the reproductive system in previous 14-day preliminary toxicity, OECD 407 (28-day toxicity) and OECD 421 (reproductive toxicity screening) studies in which oral (gavage) administration was employed.
- Post-exposure recovery period in satellite groups: 4 weeks (28 days)

EXAMINATIONS:

CAGE SIDE OBSERVATIONS: Yes , at least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes. Performed on each animal on Days -3, 1, 4, 8, 11, 15 and 19 of the study to monitor general health. In addition, recovery phase animals were assessed weekly following cessation of treatment.

BODY WEIGHT: Yes. Recorded before treatment commenced (Day -3), on Day 1, and on Days 4, 8, 11, 15 and 19 (before necropsy for main phase animals). Following cessation of treatment, Recovery phase animals were weighed Days 1, 4, 8, 11, 15, 19, 22, and 29 (before necropsy) of recovery.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, the weight of food supplied to each cage, that remaining and an estimate of any food spilled was recorded before treatment (Day -3 to -1) and twice weekly during treatment (Days 1-3, 4-7, 8-10, 11-14, and 15-18). Recordings were made weekly for Recovery phase animals following cessation of treatment.

FOOD EFFICIENCY: Not assessed.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes (assessed by daily visual observation; as no effects were observed, quantitative measurements were not performed)

OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No

ORGAN WEIGHTS: Yes
GROSS PATHOLOGY: Yes (all tissues)
HISTOPATHOLOGY: Yes (specific organs: right epididymides, right testes, tissue abnormalities (if any))

Statistics:

The following data types were analysed at each time point separately:
-- Body weight, using gains over appropriate study periods
-- Organ weights, absolute and adjusted for terminal body weight
-- Sperm analysis

A parametric analysis was performed for organ weight data and sperm analysis if Bartlett's test for variance homogeneity was not significant at the 1% level. For pre-treatment data, analysis of variance was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using t-tests, with the error mean square from the one-way analysis of variance, were made. For all other comparisons the F1 approximate test was applied.

A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. For pre treatment data, Kruskal-Wallis’ test was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using Wilcoxon rank sum tests were made. For all other comparisons, the H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied.

For sperm analysis data, if 75% of the data (across all groups) were the same value, for example c, Fisher’s Exact tests were performed.

For organ weight data, analysis of covariance was performed using terminal body weight as covariate, unless non-parametric methods were applied.

Dose descriptor:

other: no effect levels were reported

Basis for effect level:

other: No effect levels were reported, given the exploratory nature of this study.

Remarks on result:

not measured/tested

Remarks:

Effect level not specified (migrated information)

Reproductive toxicity-related data are provided here; other organ weight, body weight, and food consumption data are provided below.

RESULTS OF SPERM ANALYSIS:

Computer assisted sperm analysis conducted after 18 days of treatment revealed a dose related reduction in sperm velocity and amplitude of lateral head displacement at 7500 ppm when compared to Controls. These differences were associated with effects on sperm motility to differing severities in three of five males [an increase in the percentage of static sperm and a marked decrease in the percentage of motile sperm (two males) and a slight (one male) or more marked (two males) decrease in progressively motile sperm] and an absence of motile and/or progressively motile sperm in the remaining two males. These effects resulted in an overall group mean of 0.48-times the Control value and 0.27-times the Control value for motile and progressively motile sperm, respectively. These changes indicate an effect on the vigour and pattern of sperm motion. There was also a suggestion of slightly low cauda epididymal weight, epididymal spermatid concentrations (millions/g, notably influenced by a single male) and total epididymal sperm number.

At 5000 ppm, one of the five males showed low percentages of motile and progressively motile sperm, with a concomitant increase in the percentage of static sperm. Consequently, the overall group mean values for these parameters were 0.88X, 0.80X and 2.83X Control, respectively. There was also a slight statistically significant decrease in sperm velocity parameters at this inclusion level as mean average path velocity (VAP) and curvilinear velocity (VCL) values were 0.81X and 0.82X of Control, respectively. While not statistically significant, mean straight line velocity (VSL) values were 0.75X of Control.

Sperm motility was considered unaffected by treatment at 1500 or 3000 ppm. It was noted that one male receiving 1500 ppm showed low sperm motility, however in the absence of effects in the 3000 ppm group, this isolated incidence was considered fortuitous and unrelated to treatment.

There was no evidence of an effect of treatment on testicular spermatid concentrations (millions/g) and total sperm number at any dose level.

Four weeks after cessation of treatment with the test substance there was clear evidence of recovery but it was not complete; sperm motility and motion parameters of the treated groups were generally slightly lower or lower than those of the Controls, however no clear dose-related trend was apparent and the absolute values of most parameters were greatly improved compared to the end of treatment period, especially at the high dose of 7500 ppm (e.g., 29% vs. 78% rapid sperm motion in high dose vs. control group at the end of treatment, compared to 68% vs. 78% in high dose vs. control group after recovery). In addition testicular spermatid numbers were marginally higher in treated animals than in Control animals. This change was statistically significant at a dietary level of 7500 ppm but was considered to be within the expected normal range for male CD rats at this age.

ORGAN WEIGHTS: Reproductive Organs only: there was a suggestion of slightly low combined seminal vesicle/prostate/coagulating gland weights and epididymal weights among males at 7500 ppm, although the differences from the Control were not statistically significant. After 4 weeks of recovery, there remained a suggestion of slightly lower combined seminal vesicle/prostate/coagulating gland weights among males in the 7500 group compared to Controls (not statistically significant). This difference was attributable to 1 male with a low value; however, it is worth noting that the value was within the historical control data for this strain of rats.

GROSS PATHOLOGY: There were no treatment-related macroscopic abnormalities detected in animals at either necropsy (main phase and recovery animals).

HISTOPATHOLOGY: Following 18 days of treatment, there were no histopathological correlates for the reduced numbers/absence of motile and progressively motile sperm observed among affected males in the 5000 or 7500 ppm groups. After the 4-week recovery period, there was similarly no histopathological correlate for the generally lower numbers of motile and progressively motile sperm in the epididymis or marginally higher testicular spermatid numbers in the Recovery phase animals. There were no cell or stage-specific abnormalities noted in the seminiferous tubules, which were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within different stages. Although undocumented as a formal finding in the Study Report results, a review of the raw data motility images in the sperm analysis showed the presence of decapitate sperm.

The dose levels of 0, 1500, 3000, 5000, and 7500 ppm in the diet resulted in achieved dose levels of 0, 109, 214, 353 and 493 mg/kg/day for males and 0, 142, 210, 339, and 499 mg/kg/day for females.

Results on body weight and body weight gain, food consumption, water consumption, organ weights, gross pathology and histopathology are detailed in the cross-referenced study 7.5.1 Repeated dose toxicity: 14-day oral (dietary).002. Noteworthy findings are briefly described below, particularly organ weight and histopathology data relevant to reproduction:

BODY WEIGHT AND WEIGHT GAIN: There was a dose-related reduction in mean weight gain or mean weight loss evident between Days 1 and Day 4 of treatment in males and females receiving at 3000 ppm and above, but improvements were observed thereafter. These decreases in body weight gain had a small impact on absolute body weight. At the end of the dosing period, statistically significant decreases in absolute body weights were observed in males and females in the 7500 ppm groups (0.89- and 0.85-times the Control values for males and females, respectively). Mean body weight gain and absolute body weights of males and females at 1500 ppm was considered unaffected by treatment. Effects on weight gain were reversed during the recovery period.

FOOD CONSUMPTION AND COMPOUND INTAKE: A dose-related reduction in mean food intake was apparent during Days 1-3 of treatment for males and females at 3000 ppm and above. From Day 4 to Day 18 of treatment, there was a general improvement in food intake, however parity with Controls was not achieved. The effects on food consumption were reversible, as observed during the recovery period. The palatability of the diet was considered to be the cause of the lower food intake. The lower food consumption resulted in a lower compound intake compared to targeted doses.

ORGAN WEIGHTS: Non-Reproductive organs: there was a trend towards slightly increased adjusted liver weight among males receiving 7500 ppm and all groups of females receiving the test substance (not statistically significant); the lower body weights in these animals may have contributed to these findings. After 4 weeks of recovery, no effects on liver weight (either adjusted or absolute) were observed, including in males that had received 7500 ppm.

Conclusions:

The results of this exploratory 14-day study, which showed a lower magnitude of effects on the sperm following dietary treatment as compared to oral gavage dosing, would suggest that future toxicology studies with the substance could be conducted using dietary administration. The decrease in magnitude of effects on the adjusted liver weights also indicates that dietary administration could be employed in further toxicology studies.

Executive summary:

The objective of this investigative study was to assess the systemic toxicity potential of the test substance following dietary administration in Crl:CD (SD) rats, with particular focus on the male reproductive system, following evidence of toxicity to the male reproductive system in previous repeated dose toxicity studies (14-day preliminary toxicity (see Section 7.5.1 Repeated dose toxicity: 14-day oral (gavage).001; 28-day toxicity (OECD Testing Guideline No. 407, see Section 7.5.1 Repeated dose toxicity: 28-day oral (gavage).001) as well as the reproductive toxicity screening (OECD Testing Guideline No. 421, see Section 7.8.1 Toxicity to reproduction (oral gavage).001) study in which oral gavage administration was employed. The 14 -day dietary study demonstrated some issues with palatability of the diet, particularly for males at 3000 ppm and for males and females at 5000 or 7500 ppm, with clear reductions in food intake apparent from the commencement of treatment and persisting throughout the 18-day administration period. As a consequence, the original planned 14-day study was extended to 18 days of dosing to account for the low food intake in the first few days of the study. A recovery period was included to allow for evaluation of effects in males at the two highest dose levels. The dose levels were 0, 1500, 3000, 5000, and 7500 ppm in the diet with resulting achieved dose levels of 0, 109, 214, 353 and 493 mg/kg/day for males and 0, 142, 210, 339, and 499 mg/kg/day for females.

Findings in this study related to the general systemic effects of the substance following 14 days of dietary administration are detailed in the summary found in Section 7.5.1 Repeated dose toxicity: 14 -day oral (dietary).002. Briefly, decreases in body weight gain and food consumption were observed in the study, particularly on Days 1 to 4 of the study, but continuing in all treated groups for the remainder of the administration period. These effects were likely related to palatability of the food and were observed to improve after the first few days, and be reversible after the cessation of treatment. There were no treatment-related macroscopic or microscopic findings in any dose group, and the organ weight data (other than those related to reproductive tissues) showed only a slight increase in adjusted liver weights in males at 7500 ppm and in groups of treated females, although no change in absolute liver weights, at the end of the dosing period (main phase animals); a similar increase was not seen following the 4 -week recovery period.

Effects on reproductive tissues are limited to observations of some reductions in sperm motility among males receiving 5000 or 7500 ppm (equivalent doses 353 and 493 mg/kg/day). Some effects on the vigour and pattern of sperm motion were evident in all males at 7500 ppm, but in only one of the five males at 5000 ppm. The inclusion of recovery groups of males in the study at the two highest dose levels showed that partial recovery of all of the sperm motility changes was apparent four weeks after the cessation of treatment. While effects on sperm vigour were observed, microscopic pathology showed that there were no histopathological correlates for these changes, including a lack of cell or spermatogenic stage-specific abnormalities following evaluation of the seminiferous tubules.

The spectrum of effects observed on the sperm motility parameters in this dietary study were consistent with those seen on the previous 14-day oral gavage study at dose levels of 300 or 500 mg/kg/day, the 28-day study at 300 mg/kg/day and in the oral gavage reproductive toxicity screening study after approximately seven weeks of treatment at 300 mg/kg/day. The magnitude of the effects observed at 5000 or 7500 ppm in the diet (corresponding to approximately 353 and 493 mg/kg/day in males, respectively) were, however, slightly less than observed in the previous studies at equivalent oral gavage doses. In all studies irrespective of the route of administration there were no microscopic correlates with the changes in sperm motility. In view of the absence of any histopathological changes in the testes and epididymides, the aetiology of the sperm effects cannot be ascertained. The data from recovery group animals after the 4-week non-dosed period indicate that the effects on the sperm were reversible and, given sufficient time post-dose, complete recovery might have been observed in previously-dosed animals in this or other studies with this substance.

Taking into account both the non-degenerative effects on the liver, which were reduced with the dietary administration compared to previous results with oral gavage dosing [see Section 7.5.1 Repeated dose toxicity: 14 -day oral (dietary).002] and on the sperm, the results of this study suggest that future toxicology studies for the substance could be conducted using dietary administration.

Reference 2

Endpoint:: toxicity to reproduction: other studies
Type of information:: experimental study
Adequacy of study:: supporting study
Study period:: January 9, 2007 to March 4, 2010
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:: OECD 421 deviation: Only 5 males per group and only 2 dose groups evaluated (50 or 1,000 mg/kg bw/day)
Qualifier:: equivalent or similar to guideline
Guideline:: other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:: yes
Remarks:: Only 5 males per group and only 2 dose groups evaluated (50 or 1,000 mg/kg bw/day). As this was a screening study designed to evaluate testicular effects, mating was not included.
Principles of method if other than guideline:: Screening study for testicular toxicity in rodents; animals were not mated.
GLP compliance:: no
Remarks:: (The study was carried out in accordance with OECD GLP practices; however, the protocol and experimental phase was not checked by the Quality Assurance Unit and a full study report was not compiled, thus, the study does not have a GLP status. )
Type of method:: in vivo
Species:: rat
Strain:: Wistar
Sex:: male
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: Charles River Laboratories, Germany
- Age at study initiation: 13-14 weeks
- Weight at study initiation: Not reported
- Fasting period before study: Not reported
- Housing: Individually in Makrolon cages type M III
- Diet (e.g. ad libitum): ad libitum, Ground Kliba maintenance diet
- Water (e.g. ad libitum): ad libitum
- Acclimation period: yes (duration not reported)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: January 9, 2007 to January 31, 2007 for 1000 mg/kg bw/day dose group or January 23, 2007 to February 12, 2007.
Route of administration:: oral: gavage
Vehicle:: olive oil
Remarks:: volume: 4 mL/kg body weight
Details on exposure:: PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): not reported
- Concentration in vehicle: 25 g/100 mL (for 1000 mg/kg bw/day dose group) or 1.25 g/100 mL (50 mg/kg bw/day dose group)
- Amount of vehicle (if gavage): volume of test substance in olive oil is 4 ml/kg body weight
- Lot/batch no. (if required): not reported
- Purity: not reported
Analytical verification of doses or concentrations:: no
Duration of treatment / exposure:: 14 days, as planned.
Frequency of treatment:: Animals were dosed daily.
Duration of test:: 14 days, as planned.
Dose / conc.:: 50 mg/kg bw/day (actual dose received)
Dose / conc.:: 1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:: 5 (males only)
Control animals:: yes, concurrent vehicle
Details on study design:: - Dose selection rationale: not reported
- Rationale for animal assignment (if not random): random
Dose descriptor:: NOAEL
Basis for effect level:: other: No NOAEL identified
Remarks on result:: not determinable
Remarks:: no NOAEL identified
Conclusions:: Based on this screening study, the substance showed the potential for adverse effects on male reproductive organs; however, further testing is recommended.
Executive summary:: In this screening study designed to evaluate potential for effects on male reproductive organs, 5 male rats per group were dosed daily with the substance at 50 or 1,000 mg/kg bw/day. While clinical signs at the low dose were limited to salivation after dosing in 1 of 5 animals on some days, mortality and signs of toxicity were observed at the high dose. Based on histopathological observations of oligospermia in the epididymis and testicular degeneration at 1,000 mg/kg bw/day, the authors determined that the data revealed the potential for adverse effects on male reproductive organs. In the 50 mg/kg bw/day group, significantly reduced total spermatids/g testis (-19%, or 95 +/- 19.3% vs. 117 +/- 13.3% in controls) was observed. While the authors observed a lower % normal sperm (-3%; or 91.1 +/- 3.2% vs. 94.0 +/- 1.3% in controls), this is noted to be a small magnitude of change and unlikely to be biologically significant. There was no statistically significant difference in total sperm/g cauda epididymis, % abnormal sperm, or % motility. It is unclear what role the state of the animals (noting clinical signs and toxicity, including mortality) may have played in the epididymal oligospermia observed at 1,000 mg/kg bw/day. As this was a screening study for toxicity to the testes only, the potential for male reproductive organ toxicity should be further evaluated.

Additional information

Summary of Data of 'Other Studies'

In the screening study designed to evaluate potential for effects on male reproductive organs, 5 male rats per group were dosed daily with the test substance at 50 or 1,000 mg/kg bw/day via oral gavage. While clinical signs at the low dose were limited to salivation after dosing in 1 of 5 animals on some days, mortality and signs of toxicity were observed at the high dose. Based on histopathological observations of oligospermia in the epididymis and testicular degeneration at 1,000 mg/kg bw/day, the authors determined that the substance may cause significant adverse effects on male reproductive organs. It is unclear what role the state of the animals (noting clinical signs) may have played in the epididymal oligospermia observed at 1,000 mg/kg bw/day. In the 50 mg/kg bw/day group, significantly reduced total spermatids/g testis (-19%, or 95 +/- 19.3% vs. 117 +/- 13.3% in controls) was observed. While the authors observed a lower % normal sperm (-3%; or 91.1 +/- 3.2% vs. 94.0 +/- 1.3% in controls), this is noted to be a small magnitude of change and unlikely to be biologically significant. There was no statistically significant difference in total sperm/g cauda epididymis, % abnormal sperm, or % motility. As this was an early screening study to evaluate toxicity to the testes only, it was considered that the potential for male reproductive organ toxicity should be further evaluated.

While both male and female rats were included in an exploratory study designed to evaluate the potential for effects following dietary dosing, the study focused on the evaluation of sperm effects in males. Five (5) male rats per group were dosed daily with the test substance at dose levels of 0 (control), 1500, 3000, 5000, and 7500 ppm (equivalent to approximately 0, 109, 214, 353, and 493 mg/kg/day) for a total of 18 days (dosing period extended to ensure at least 14 days of exposure to the substance at maximum dose levels). Sperm analysis showed effects on the vigour and pattern of sperm motion at 7500 ppm, with the data also suggesting slightly low cauda epididymal weight, epididymal spermatid concentrations (influenced by a single male) and total epididymal sperm number. At 5000 ppm, one of five males showed similar effects on sperm vigour. However, partial recovery of all of the sperm motility changes was apparent four weeks after the cessation of treatment. Moreover, microscopic pathology showed that there were no histopathological correlates for the effects on sperm vigour at the end of the 18-day treatment period or at the end of the 4-week recovery period, including a lack of cell or spermatogenic stage-specific abnormalities. Based on the results of this investigative study, it was concluded that the treatment-related effects observed on the sperm of male rats exposed to the substance by oral gavage dosing were slightly reduced by employing dietary administration. The lower magnitude of sperm effects in this dietary study compared to that seen after oral gavage dosing would suggest that future preclinical studies with this substance could be conducted using dietary administration.

Justification for classification or non-classification

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, 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.

As the substance does not display intrinsic reproductive toxicity (either to parental animals or to pups) at the dose level of 300 mg/kg/day, based on the screening study and developmental toxicity study, and in consideration of the scientific arguments that sperm-related effects may have been the result of conditions of oxidative stress following oral gavage administration of a high dose level, it is considered that there is insufficient evidence for classification at this time according to Regulation (EC) No 1272/2008, Annex I section 3.9.

Additional information

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Registration Dossier

Registration Dossier

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Diss Factsheets

Cyclohexanemethanol, 4-(1-methylethyl)-

Endpoint summary

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Link to relevant study records

Reference

Effect on fertility: via oral route

Effect on fertility: via inhalation route

Effect on fertility: via dermal route

Additional information

Effects on developmental toxicity

Description of key information

Link to relevant study records

Reference

Effect on developmental toxicity: via oral route

Effect on developmental toxicity: via inhalation route

Effect on developmental toxicity: via dermal route

Additional information

Toxicity to reproduction: other studies

Description of key information

Link to relevant study records

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Reference 1

Reference 2

Additional information

Justification for classification or non-classification

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