Reaction mass of 2-(cis-4-methylcyclohexyl)propan-2-ol and 2-(trans-4-methylcyclohexyl)propan-2-ol and cis-1-methyl-4-(propan-2-yl)cyclohexanol and trans-1-methyl-4-(propan-2-yl)cyclohexanol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted in rats according to OECD Guideline 422 and in compliance with GLP, no NOAEL could be identified as no adverse toxic effects were observed at the highest dose tested, i.e. 15000 ppm (representing mean achieved dose levels of 944 mg/kg bw/day for males and 1065 and 1874 mg/kg bw/day for reproductive phase females during gestation and lactation, respectively).

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 December 2013 to 17 October 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

November 2015

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited
- Age of F0 animals at study initiation: Approximately 10 weeks
- Weight at study initiation: Males: 340-397 g; Females: 224-273 g
- Housing: No. of animals per cage - Males: pre pairing and post pairing - up to 5 animals; Females: pre pairing - up to 5 animals; During pairing - one male and one female; Gestation - one female; Lactation: one female + litter. Toxicity phase and recovery phase - up to five animals of one sex. Cages comprised of a polycarbonate body with a stainless steel mesh lid. Solid (polycarbonate) bottom cages were used during the acclimatisation, gestation, littering and lactation periods. Grid bottomed 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

ENVIRONMENTAL CONDITIONS
- Temperature: 19-23 °C
- Humidity: 40-70 %
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: 12 h dark / 12 h light

Route of administration:

oral: feed

Vehicle:

other: carrier diet, including corn oil

Details on exposure:

DIET PREPARATION
- Carrier diet: SDS VRF1 certified diet.
- Stabiliser: Corn oil (test material to corn oil ratio 5:1)
- Method of preparation: On each occasion of the preparation of the premix the required amount of test substance and corn oil were mixed together and stirred. The mixture was added to an equal amount of plain diet and stirred until visibly homogenous. This doubling up process was repeated until half of the final weight of premix was achieved or the mixture appeared dry. This mixture was then ground using a mechanical grinder after which it was made up to the final weight of the premix with plain diet. This premix was mixed in a Turbula mixer for 100 cycles to ensure the test substance was dispersed in the diet. Aliquots of the premix were then diluted with further quantities of plain diet to produce the required dietary concentrations. Each batch of treated diet was mixed for a further 100 cycles in a Turbula mixer.
For Control diet the corn oil was added directly to the diet and then prepared as indicated for the premix.
- Frequency of preparation: Weekly
- Storage of preparation: Frozen (nominally -20 °C).

GROUPS
Groups 1, 2, 3 and 4: 0, 1500, 5000 and 15000 ppm, respectively

Details on mating procedure:

- Toxicity phase and recovery phase males were paired with reproductive phase females. Toxicity and recovery phase females were not paired for mating.
- M/F ratio per cage: 1:1 from within the same treatment groups
- Pairing commenced: After three weeks of treatment
- Length of cohabitation: Up to 2 weeks
- Proof of pregnancy: Presence of sperm within the vaginal smear and/or ejected copulation plugs referred to as Day 0 of pregnancy.
- Male/female separation: Day when mating evidence was detected.
- Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 100 and 20000 ppm were analysed to assess the stability and homogeneity of the test substance in the diet matrix.
Stability was confirmed at two days ambient temperature and 22 days when stored frozen at 100 ppm. Stability was confirmed at eight days ambient temperature and 22 days when stored frozen at 20000 ppm.
Diet in hoppers was changed daily.
Achieved concentration: Samples of each formulation prepared for administration in Weeks 1 and 5 of treatment were analysed for achieved concentration of the test substance.

Duration of treatment / exposure:

Toxicity phase males were treated daily for three weeks before pairing, throughout pairing and up to necropsy after a minimum of five consecutive weeks.
Toxicity phase females were treated daily for a minimum of five consecutive weeks up to necropsy in Week 6.
Reproductive females were treated daily for three weeks before pairing, throughout pairing, gestation and until Day 6 of lactation.
Five high dose Recovery phase male and female rats were treated daily for a minimum of six consecutive weeks, followed by a 14 day period without treatment.

Frequency of treatment:

Continuously (fresh diet given daily). During the recovery period, all animals were given untreated diet (without corn oil).

Dose / conc.:

15 000 ppm

Remarks:

nominal in diet

Dose / conc.:

1 500 ppm

Remarks:

nominal in diet

Dose / conc.:

5 000 ppm

Remarks:

nominal in diet

No. of animals per sex per dose:

Reproduction phase: 10 females/dose
Toxicity phase : 5 animals/sex/dose for vehicle and high dose groups; 10 males/dose for 1500 and 5000 ppm dose groups; 5 females/dose for 1500 and 5000 ppm dose groups
Recovery phase: 5 for vehicle and high dose groups

Control animals:

other: control group was provided with the carrier diet, including corn oil

Details on study design:

- Dose selection rationale: Dose levels were selected in conjunction with the Sponsor following the completion of the dose range finder study (Study number OAD0015). Dose levels were selected as 1500, 5000 and 15000 ppm. In the dose range finding study the doses of 1500, 7500 or 15000 ppm were well tolerated, no adverse clinical signs or macroscopic changes in organ appearance were detected at any dose level. Effects of treatment at 15000 ppm included initial body weight loss and low food consumption for up to four days following the start of dosing; liver weights were also high in these animals.
- Rationale for animal assignment: Randomly allocated on arrival. 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.

Positive control:

Not applicable

Parental animals: Observations and examinations:

CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced and during each week of treatment and recovery, detailed physical examination and arena observations were performed on each adult animal. In addition, detailed physical examination and arena observations were performed on reproductive phase females on Days 0, 6, 13 and 20 after mating and Days 1 and 6 of lactation. On each occasion, the examinations were performed at approximately the same time of day, by an observer unaware of the experimental group identities.
During the acclimatisation and recovery periods, observations of the animals and their cages were recorded at least once per day.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Before treatment commenced and during each week of treatment and recovery, detailed physical examination and arena observations were performed on each animal.
- Time schedule:
- Sensory reactivity and grip strength: Sensory reactivity and grip strength assessments were performed on all recovery animals in Groups 1 and 4 and the five lowest numbered surviving toxicity phase males and females in Groups 2 and 3, during Week 6 of treatment.
- Motor activity:
During Week 6 of treatment, the motor activity of all recovery animals in Groups 1 and 4 and the five lowest numbered surviving toxicity phase males and females in Groups 2 and 3 was measured.

BODY WEIGHT: Yes
- Time schedule for examinations:
Toxicity and Recovery phase animals: Before feeding on the day that treatment commenced (Day 1), weekly thereafter and before necropsy.
Reproductive females: Before feeding on the day that treatment commenced (Day 1), weekly before pairing, on Days 0, 6, 13 and 20 after mating, on Days 1, 4 and 7 of lactation and before necropsy.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: The weight of food supplied, that remaining and an estimate of any spilled was recorded daily for all animals. From these records the mean daily consumption per animal (g/animal/day) was calculated for each phase. Food consumption was not recorded for males and females during the period when paired for mating (Day 22 to 28). Food consumption recordings recommenced on Day 29.


PARTURITION OBSERVATIONS AND GESTATION LENGTH:
- Duration of gestation: Time elapsing between the detection of mating and commencement of parturition.
- 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.

Oestrous cyclicity (parental animals):

- Dry smears: Daily smears were taken for 15 days before pairing, using cotton swabs moistened with saline. 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.

Litter observations:

PARAMETERS EXAMINED
- Clinical observations: Examined at approximately 24 h 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.
- Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1-7 of age.
- Sex ratio: The sex ratio of each litter was recorded on Days 1, 4 and 7 of age.
- Individual offspring bodyweights: F1 offspring - Days 1, 4 and 7 of age.

Postmortem examinations (parental animals):

SACRIFICE
- Reproductive phase females: Scheduled kill - Day 7 of lactation; Failing to produce viable litter - Day 25 after mating.
- Recovery phase animals: After at least 14 days without treatment.
- Method of sacrifice: F0 animals were killed by Carbon dioxide asphyxiation with subsequent exsanguination.

GROSS NECROPSY
- All adult animals were subject to a detailed necropsy. 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. In addition the number of uterine implantation sites recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
For bilateral organs, left and right organs were weighed together.
Histology
Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
The five lowest numbered males and females in Groups 1 and 4 at scheduled termination.
Abnormalities only: All animals
Kidneys: All males from the toxicity (Group 2 and 3) and recovery (Group 1 and 4) phases which had tissues retained.
- Routine staining: Sections were stained with haematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid Schiff (PAS) method.
- Tissues were routinely preserved in 10 % Neutral Buffered Formalin with the exception of Testes in modified Davidson’s fluid; Eyes In Davidson’s fluid.
Light microscopy
- Terminal sacrifice:
Groups 1 and 4: Five lowest numbered F0 males and five Toxicity phase F0 females - All specified in table 7.8.1/1
All adult animals from all groups/phases - Abnormalities

Postmortem examinations (offspring):

SACRIFICE:
- F1 offspring scheduled kill: Day 7 of age
- Method of sacrifice: Intraperitoneal injection of sodium pentobarbitone

GROSS NECROPSY
- Offspring at scheduled termination: All F1 offspring were examined externally; any offspring found to be externally abnormal were also examined internally.
- 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 subject to fresh macroscopic examination, where possible (external and internal); this also included an assessment for the presence of milk in the stomach, where this was possible. Abnormal tissues retained.

Statistics:

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

Reproductive indices:

Mating Performance and Fertility:
- Percentage mating = (Number animals mating / Animals paired) x 100
- Conception rate (%) = (Number animals achieving pregnancy / Animals mated) x 100
- Fertility index (%) = (Number animals achieving pregnancy / Animals pairing) x 100

Gestation length
- Gestation index (%) = (Number of live litters born / Number pregnant) x 100

Offspring viability indices:

Survival indices:
- Post-implantation survival index (%) = (Total number of offspring born / Total number of uterine implantation sites) x 100
- Live birth index (%) = (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100
- Lactation index (%) = (Number of live offspring on Day 7 after littering / Number live offspring on Day 4) x 100

Sex ratio:
- Percentage males = (Number of males in litter / Total number of offspring in litter) x 100

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The bodyweight change of males receiving 15000 ppm was significantly lower than Control for the first week of study. This was accompanied by a period of low food consumption which was attributed to the animals becoming accustomed to the diet. Thereafter there was no notable difference in body weight gain to the Control although for the 3 week pre-pairing period and the 6 week treatment period, the overall weight gain of the males receiving 15000 ppm remained significantly lower than Control.
During gestation, weight gain of females receiving 15000 ppm was generally lower than Control, attaining statistical significance by Day 20 of gestation for absolute bodyweights and the period Days 0-20 for bodyweight change, mainly due to a decrease in body weight gain during Days 13-20. As a consequence, bodyweights of females in the 15000 ppm group were statistically lower than Control on Day 1 of lactation; this statistical difference was also present on Day 7 of lactation. However, bodyweight change during lactation did not indicate a clear effect of treatment at any dose level.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Short lasting reduction in food consumption was observed between day 1 and 4, in animals receiving 15000 ppm but was attributed to the low palatability of the preparation and is not considered to be adverse.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Blood urea concentration was higher, attaining statistical significance, compared with Control with an indication of a dose trend. However, urea concentration was within historical control data (historical control 90 percentile range : 4.72-6.60 mmol/L, n=20, year 2014-2015).
Chloride concentration was significantly lower than control in males receiving 15000 or 5000 ppm but stayed in historical control data (historical control 90 percentile range : 98-103 mmol/L, n=20, year 2014-2015).
Other changes detected at the week 6 sampling occasion included bile acid concentration lower than controls in males at the low and intermediate dose levels only however the highest concentration group was similar to control. It was not accompagnied by histopathological findings.
Calcium was significantly higher than Control in males at 5000 and 15000 ppm. No other mineral concentration was modificated. After 14 days without the test material there were no significant differences to Control in all previously affected parameters after 14 days off dose. It was noted that calcium in males was marginally but significantly lower than Control but this was considered to have arisen by chance.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Animals killed after 5 weeks of treatment
Changes related to treatment with Dihydroterpineol multiconstituent were seen in kidneys of male animals.
Hyaline droplets in the cortical tubules were seen in all male treated groups with corticomedullary granular casts seen in males given 5000 and 15000 ppm of Dihydroterpineol multiconstituent. An increase incidence and severity of cortical tubular basophilia was seen in males given
5000 and 15000 ppm of Dihydroterpineol multiconstituent.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- The profile of clinical signs did not indicate any reaction to the test item.


BODY WEIGHT (PARENTAL ANIMALS)
- There was no clear indication of an effect of the test item on males receiving 5000 or 1500 ppm. Changes detected at 15000 ppm in males, though attributed to treatment, were considered not to be adverse.
- The bodyweight change of males receiving 15000 ppm was significantly lower than Control for the first week of study. This was accompanied by a period of low food consumption which was attributed to the animals becoming accustomed to the diet. Thereafter there was no notable difference in bodyweight gain to the Control although for the 3 week pre-pairing period and the 6 week treatment period, the overall weight gain of the males receiving 15000 ppm remained significantly lower than Control.
- During gestation, weight gain of females receiving 15000 ppm was generally lower than Control, attaining statistical significance by Day 20 of gestation for absolute bodyweights and the period Days 0-20 for bodyweight change, entirely due to a decrease in bodyweight gain during Days 13-20. As a consequence, bodyweights of females in the 15000 ppm group were statistically lower than Control on Day 1 of lactation; this statistical difference was also present on Day 7 of lactation. However, bodyweight change during lactation did not indicate a clear effect of treatment at any dose level.

FOOD CONSUMPTION (PARENTAL ANIMALS)
- Food consumption of animals receiving 15000 ppm on Day 1 of study was markedly lower than Control. Intake increased each day and by Day 4 of dosing there was no notable effect on food consumption at any dose level. This short lasting reduction in food consumption is attributed to the low palatability of the preparation and is not considered to be adverse.
- Food consumption for females during gestation was not affected by Dihydroterpineol multiconstituent. The periods 0-5 and 6-12 were statistically lower than Control these minor differences were not considered to be adverse.
- During lactation at 15000 ppm, food consumption gradually diverged from the Control so that by Day 5 of lactation the reduction in comparison to Control attained statistical significance.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Before pairing (all animals) and after pairing (toxicity and recovery animals): 106, 342 and 971 mg/kg bw/day for 1500, 5000 and 15000 ppm, respectively in females; 96, 314 and 944 mg/kg bw/day for 1500, 5000 and 15000 ppm, respectively in males
Gestation: 108, 351 and 1065 mg/kg bw/day for 1500, 5000 and 15000 ppm, respectively in females
Lactation: 189, 663 and 1874 mg/kg bw/day for 1500, 5000 and 15000 ppm, respectively in females
- Achieved dose generally maintained the ratio between dose levels. As expected, with the increased physiological demand, achieved dose increased slightly during gestation and significantly during lactation.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- There was no effect of the test item on oestrous cycles pre-coital interval, mating performance, fertility, gestation length or gestation index.

ORGAN WEIGHTS (PARENTAL ANIMALS)
- Liver weight for toxicity phase males and females receiving 15000 ppm were statistically higher than Control, this was also apparent in males only at 5000 ppm; there was also an indication of a similar effect in the reproductive phase females but not attaining statistical significance. Toxicity phase males, females and reproductive phase females receiving 15000 ppm had high adrenal weights in comparison with Control; the difference did not attain statistical significance for toxicity phase females.
- Marginally but statistically significant high ovary weights were recorded only for the reproductive phase females receiving 15000 ppm; this change was not considered to be adverse at the degree observed. Low thymus weight recorded for toxicity phase females at all levels did not demonstrate a dose response and was considered not to be an adverse effect of treatment.
After 14 days of dose, none of the parameters affected above were significantly different to the Control. Males which had previously received 15000 ppm had high spleen weights in comparison to recovery Control but they had lower spleen weights when compared with toxicity phase Control. Females at this dose level had slightly but significantly lower kidney weights.

GROSS PATHOLOGY (PARENTAL ANIMALS)
- There was no indication of any reaction to the test item in the profile of macroscopic changes observed in toxicity phase males and females, reproductive phase females or recovery phase males and females.

HISTOPATHOLOGY (PARENTAL ANIMALS)
- Animals killed after 5 weeks of treatment: Changes related to treatment with Dihydroterpineol multiconstituent were seen in kidneys of male animals. Hyaline droplets in the cortical tubules were seen in all male treated groups with corticomedullary granular casts seen in males given 5000 and 15000 ppm of Dihydroterpineol multiconstituent. An increase incidence and severity of cortical tubular basophilia was seen in males given 5000 and 15000 ppm of Dihydroterpineol multiconstituent.
- Incidental findings: An adenocarcinoma was recorded in the mammary gland of a female given 1500 ppm of Dihydroterpineol multiconstituent. This was considered to be incidental. All other histological changes were considered to be unrelated to treatment.
- Animals killed after 14 days of recovery: Changes related to treatment with Dihydroterpineol multiconstituent were seen in kidneys of male recovery animals. The findings of cortical tubular basophilia and corticomedullary granular casts, seen in male terminal animals, were also present in the recovery animals previously treated with 15000 ppm of Dihydroterpineol multiconstituent. Hyaline droplets in the cortical tubules, seen in terminal animals, were not seen in recovery animals.
Cortical tubular basophilia, although present in all previously treated animals, was present at a minimal grade only which may indicate partial recovery. The granular casts were seen at a slightly reduced incidence in the recovery animals previously treated with 15000 ppm compared to terminal animals which may indicate partial recovery.

OTHER FINDINGS (PARENTAL ANIMALS)
Haematology: There were no effects of test item on any of the haematology parameters assessed.
Blood chemistry: Analysis of serum after six weeks of exposure indicated high urea and low chloride concentrations in animals receiving 15000 ppm. Bile acid was significantly lower than control in males at the intermediate and low dose. Cholesterol was higher than Control for males receiving 15000 ppm with an indication of a dose trend at 5000 ppm in males. Calcium was significantly higher than Control in males at 15000, and at 5000 ppm.

Sensory reactivity and grip strength: There was no effect of test item on sensory reactivity or grip strength.
Motor activity: No effect of treatment on motor activity in males or females at dose levels up to 15000 ppm. There were two statistically significant periods of high low beam activity (at 54 minutes and 60 minutes) above the Control for females at 15000 ppm. This difference was noted for females only and group mean values were within the historical Control range.

Key result

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Highest dose level tested, corresponding to 944 mg/kg bw/day for males and 1065 and 1874 mg/kg bw/day for reproductive phase females during gestation and lactation, respectively.

Critical effects observed:

yes

Lowest effective dose / conc.:

5 000 ppm

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The only reproductive endpoint considered to be affected by treatment was offspring body weight gain which was low for litters derived from parents receiving 15000 ppm compared with the Control. This did not have any impact on the survival or clinical condition of the offspring and therefore, within the context of this study, it was considered not to be adverse.

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 specified

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

CLINICAL SIGNS (OFFSPRING)
There were no changes in clinical condition of the offspring which indicated a reaction to parental exposure to test item.

LITTER SIZE, SURVIVAL INDICES AND SEX RATIO
Survival, litter size and sex ratio were not adversely affected by parental exposure to test item.

BODY WEIGHT (OFFSPRING)
There was no difference to Control in offspring bodyweight on Day 1 of age; growth thereafter was lower in male and female offspring from parents receiving 15000 ppm. There was no effect of treatment in offspring from parents receiving 5000 or 1500 ppm.

GROSS PATHOLOGY (OFFSPRING)
There were no changes detected at examination of the offspring dying before or at scheduled termination which indication any effect of the test item.

Key result

Generation:

F1

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Formulation analysis:

The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limit of detection, linearity of detector response, repeatability, accuracy and precision.

The homogeneity was confirmed for Dihydroterpineol multiconstituent in SDS VRF1 certified diet formulations at nominal concentrations of 100 ppm and 20000 ppm. Stability was confirmed at ambient storage for 2 days and 8 days at 100 ppm and 20000 ppm, respectively. Stability was confirmed at frozen storage up to 22 days at 100 ppm and 20000 ppm.

The mean concentration of Dihydroterpineol multiconstituent in each test formulations analysed for the study was within +10%/-15% of nominal concentrations, confirming accurate formulation. The percent difference from mean remained less than 3% confirming the accuracy of analysis.

Conclusions:

No NOAEL for this six week repeat dose toxicity study with 14-day recovery period including a reproductive screen/developmental toxicity could be identified as no adverse toxic effects were observed at the highest dose tested, i.e. 15000 ppm (representing mean achieved dose levels of 944 mg/kg bw/day for males and 1065 and 1874 mg/kg bw/day for reproductive phase females during gestation and lactation, respectively).

Executive summary:

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted according to OECD Guideline 422 and in compliance with GLP, groups of Crl:CD(SD) rats received dihydroterpineol multiconstituent orally, via the diet, at concentrations of 1500, 5000 or15000 ppm. Toxicity phase males were treated daily for three weeks before pairing, throughout pairing and up to necropsy after a minimum of five consecutive weeks. Toxicity phase females were treated daily for a minimum of five consecutive weeks up to necropsy in Week 6. Reproductive females were treated daily for three weeks before pairing, throughout pairing, gestation and until Day 6 of lactation. Five high dose Recovery phase male and female rats were treated daily for a minimum of six consecutive weeks, followed by a 14-day period without treatment. Recovery phase males were used for pairing with reproductive phase females, but recovery females were not paired.

During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, haematology, blood chemistry, oestrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic investigations were undertaken for all adult animals. Microscopic pathology investigations were undertaken in the first five Toxicity phase males and Reproductive in Group 1 (Control) and Group 4 (15000 ppm). The clinical condition of offspring, litter size and survival, sex ratio and bodyweight were assessed, and macroscopic pathology investigations were undertaken. The F1 generation received no direct administration of the test substance; any exposure was in utero or via the milk.

The mean concentrations of dihydroterpineol multiconstituent in formulations were within applied limits of +10%/-15%, confirming the accuracy of formulation. The difference from mean values was <3%, confirming precise analysis. With the exception of one recovery in Week 1, procedural recovery values remained within 96.7 and 103.0%, confirming the continued accuracy of the method.

There were no adverse effects attributed to treatment on clinical condition, sensory reaction, grip strength, motor activity, haematology and macroscopic pathology. In addition the reproductive endpoints assessed which were unaffected by treatment included oestrous cycles, pre-coital interval, mating performance, fertility, gestation length, offspring clinical condition, litter size, survival sex ratio or external development. 

During gestation body weight gain was slightly lower than Control in females receiving 15000 ppm; this was reflected in low absolute body weights for these animals during lactation. Food consumption during lactation was also slightly low, in comparison to the Control. 

Analysis of serum after six weeks of exposure indicated high urea and low chloride concentrations in animals receiving 15000 ppm. Bile acid was significantly lower than control in males at the intermediate and low dose. Cholesterol was higher than Control for males receiving 15000 ppm with an indication of a dose trend at 5000 ppm in males. Calcium was significantly higher than Control in males at 15000 and at 5000 ppm. At analysis during recovery week two, none of the above changes were apparent. 

At necropsy the liver and adrenal weights for males at 15000 ppm and males at 5000 ppm (liver only) were higher than Control. After 14 days recovery these differences were not apparent.

Findings seen in the kidneys of male animals comprised of hyaline droplets in the cortical tubules, cortical tubular basophilia and corticomedullary granular casts, all at generally minimal or slight severity. Hyaline droplet formation was seen in all treated male groups after six weeks of treatment but not following 14 days of recovery. Cortical tubular basophilia and corticomedullary granular casts were seen in males given 5000 and 15000 ppm of dihydroterpineol multiconstituent. After cessation of treatment for 14 days in males given 15000 ppm there was a slight reduction in the incidence or severity of these findings, which may indicate partial recovery.

The only reproductive endpoint considered to be affected by treatment was offspring body weight gain which was low for litters derived from parents receiving 15000 ppm compared with the Control. This did not have any impact on the survival or clinical condition of the offspring and therefore, within the context of this study, it was considered not to be adverse.

Therefore, no NOAEL for this six week repeat dose toxicity study with 14-day recovery period including a reproductive screen/developmental toxicity could be identified as no adverse toxic effects were observed at the highest dose tested, i.e. 15000 ppm (representing mean achieved dose levels of 944 mg/kg bw/day for males and 1065 and 1874 mg/kg bw/day for reproductive phase females during gestation and lactation, respectively).

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted according to OECD Guideline 422 and in compliance with GLP,groups of Crl:CD(SD) ratsreceived dihydroterpineol multiconstituent orally, via the diet, at concentrations of 1500, 5000 or15000 ppm.Toxicity phase males weretreated dailyfor three weeks before pairing, throughout pairing and up to necropsy after a minimumof five consecutive weeks. Toxicity phase femaleswere treateddaily for a minimumof five consecutive weeksup to necropsy in Week 6.Reproductivefemales were treated daily for three weeks before pairing, throughoutpairing, gestation and until Day6of lactation. Five high dose Recovery phase male and female rats were treated daily for a minimumofsixconsecutive weeks, followed by a 14-day period without treatment. Recovery phase males were used for pairing with reproductive phase females, but recovery females were not paired.

During the study, clinical condition,detailed physical and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, haematology, blood chemistry,oestrous cycles, pre-coital interval, mating performance,fertility,gestation length, organ weight and macroscopic investigationswere undertaken for all adult animals. Microscopicpathology investigations were undertaken in thefirst five Toxicity phase males and Reproductive inGroup 1 (Control) andGroup4 (15000 ppm). Theclinical condition of offspring,litter size and survival, sex ratio and bodyweight were assessed,and macroscopic pathology investigations were undertaken. The F1 generation received no direct administration of the test substance; any exposure was in utero or via the milk.

The mean concentrations of dihydroterpineol multiconstituent in formulations were within applied limits of +10%/-15%, confirming the accuracy of formulation. The difference from mean values was <3%, confirming precise analysis. With the exception of one recovery in Week 1, procedural recovery values remained within 96.7 and 103.0%, confirming the continued accuracy of the method.

There were no adverse effects attributed to treatment on clinical condition, sensory reaction, grip strength, motor activity, haematology and macroscopic pathology. In addition the reproductive endpoints assessed which were unaffected by treatment included oestrous cycles, pre-coital interval, mating performance, fertility, gestation length, offspring clinical condition, litter size, survival sex ratio or external development. 

During gestation body weight gain was slightly lower than Control in females receiving 15000 ppm; this was reflected in low absolute body weights for these animals during lactation. Food consumption during lactation was also slightly low, in comparison to the Control. 

Analysis of serum after six weeks of exposure indicated high urea and low chloride concentrations in animals receiving 15000 ppm. Bile acid was significantly lower than control in males at the intermediate and low dose. Cholesterol was higher than Control for males receiving 15000 ppm with an indication of a dose trend at 5000 ppm in males. Calcium was significantly higher than Control in males at 15000 and at 5000 ppm. At analysis during recovery week two, none of the above changes were apparent. 

At necropsy the liver and adrenal weights for males at 15000 ppm and males at 5000 ppm (liver only) were higher than Control. After 14 days recovery these differences were not apparent.

Findings seen in the kidneys of male animals comprised of hyaline droplets in the cortical tubules, cortical tubular basophilia and corticomedullary granular casts, all at generally minimal or slight severity. Hyaline droplet formation was seen in all treated male groups after six weeks of treatment but not following 14 days of recovery. Cortical tubular basophilia and corticomedullary granular casts were seen in males given 5000 and 15000 ppm of dihydroterpineol multiconstituent. After cessation of treatment for 14 days in males given 15000 ppm there was a slight reduction in the incidence or severity of these findings, which may indicate partial recovery.

The only reproductive endpoint considered to be affected by treatment was offspring body weight gain which was low for litters derived from parents receiving 15000 ppm compared with the Control. This did not have any impact on the survival or clinical condition of the offspring and therefore, within the context of this study, it was considered not to be adverse.

Therefore, no NOAEL for this six week repeat dose toxicity study with 14-day recovery period including a reproductive screen/developmental toxicitycould be identified as no adverse toxic effects were observed at the highest dose tested, i.e.15000 ppm (representing mean achieved dose levels of 944 mg/kg bw/day for males and 1065 and 1874 mg/kg bw/day for reproductive phase females during gestation and lactation, respectively).

Justification for classification or non-classification

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted in rats according to OECD Guideline 422 and in compliance with GLP, no NOAEL could be identified as no adverse toxic effects were observed at the highest dose tested, i.e. 15000 ppm (representing mean achieved dose levels of 944 mg/kg bw/day for males and 1065 and 1874 mg/kg bw/day for reproductive phase females during gestation and lactation, respectively).

Therefore, dihydroterpineol multiconstituent does not need to be classified according to CLP Regulation (EC) n° 1272/2008.