Cyclohexanemethanol, 4-(1-methylethyl)-

Toxicity to reproduction: other studies

Administrative data

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

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

cis-4-(isopropyl)cyclohexanemethanol

EC Number:

237-539-8

EC Name:

cis-4-(isopropyl)cyclohexanemethanol

Cas Number:

13828-37-0

Molecular formula:

C10H20O

IUPAC Name:

cis-(4-propan-2-ylcyclohexyl)methanol

Constituent 2

Reference substance name:

trans-(4-propan-2-ylcyclohexyl)methanol

Cas Number:

13674-19-6

Molecular formula:

C10H20O

IUPAC Name:

trans-(4-propan-2-ylcyclohexyl)methanol

Test material form:

liquid

Details on test material:

- Physical state: Clear colourless liquid
- Storage condition of test material: At ambient temperature and protected from light

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 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)

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 500 ppm

Doses / concentrations 2

Dose / conc.:

3 000 ppm

Doses / concentrations 3

Dose / conc.:

5 000 ppm

Doses / concentrations 4

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.

Results and discussion

Effect levels

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)

Observed effects

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.

Any other information on results incl. tables

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. 

Applicant's summary and conclusion

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.