Menthol

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity - with F2 generation (Cohorts 1A, and 1B with extension)

Remarks:

The study was requested by the European Chemicals Agency (ECHA) decision number TPE-D-2114407364-56-01/F

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Treatment of F0 animals commenced 28 May 2019. Experimental completion date (histopathology): 26 February 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

The purpose of this study was to assess the influence of D,L-Menthol on reproductive performance when administered continuously in the diet to CD rats. The evaluation included assessment of the integrity and performance of the adult male and female reproductive tract, and systemic toxicity in pregnant and lactating females and in young and adult offspring. In addition, an evaluation of the maturing reproductive tract and its integrity and function.

Animal Model
The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Crl:CD(SD) strain was used because of the historical control data available at this laboratory.

Route of Administration
The dietary route of administration was chosen to simulate the conditions of possible human exposure

Test material

Test material information

Constituent 1

Reference substance name:

Menthol

EC Number:

201-939-0

EC Name:

Menthol

Cas Number:

89-78-1

Molecular formula:

C10H20O

IUPAC Name:

2-isopropyl-5-methylcyclohexanol

Test material form:

solid

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

Number of animals
Supplied as 54 litters of identified litter mates.
27 litters of four male siblings and 27 litters of four female siblings; no male/female sibling relationships.
Spare animals were removed from the study room after treatment commenced.

Duration of acclimatization
Six days before commencement of treatment.

Age of the F0 animals at the start of the treatment Approximately 4-5 weeks of age; (approximately 14-15 weeks of age at pairing).

Weight range of the F0 animals at the start of the treatment Males 73 to 122 g. Females 71 to 123 g.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Rodent facility
Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.

Air supply Filtered fresh air which was passed to atmosphere and not recirculated.

Temperature and relative humidity Monitored and maintained within the range of 20-24°C and 40-70%.

Although conditions were occasionally outside the indicated ranges, these deviations were minor and/or of short duration and were considered not to have influenced the health of the animals and/or the outcome of the study.

Lighting Artificial lighting, 12 hours light: 12 hours dark.

Electricity supply Public supply with automatic stand-by generators.

Animal Accommodation
Cages Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used throughout the study except during pairing.
Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily.
Cage distribution The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups.

Bedding Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.

Number of animals per cage
Acclimatization, treatment and selected F1 maturation : up to four animals of one sex
Pairing one male and one female
Males to termination up to four animals
Females after mating (from Day 0 after mating) one animal
Females during and after littering up to weaning one animal + litter
Females to termination (after weaning) up to four animals
Offspring maturation (from weaning until selection) litter

Environmental Enrichment
Aspen chew block A soft white untreated wood block; provided to each cage throughout the study (except when animals were separated into single housing overnight prior to functional observational battery testing and during lactation, or when animals were in metabolism cages for urine collection) and replaced when necessary. For F0 and F1 Cohort 1B females, chew blocks were returned on Day 21 of lactation after weaning of the offspring.

Plastic shelter Provided to each cage throughout the study (except when animals were separated into single housing overnight prior to functional observational battery testing and during lactation, or when animals were in metabolism cages for urine collection) and replaced at the same time as the cages. For F0 females and F1 Cohort 1B, shelters were returned on Day 21 of lactation after weaning of the offspring.

Paper shavings From Day 20 after mating and throughout lactation, approximately two handfuls of paper shavings were provided to each cage as nesting material; this nesting material was changed at the same frequency as the cage bedding.

Diet Supply
Diet SDS VRF1 Certified, powdered diet.
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Availability Non-restricted (diet was removed overnight before blood sampling for hematology or blood chemistry and during the period of urine collection).

Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability Non-restricted (except during urine collection).

Supplier Certificates of Analysis
Certificates of analysis for the diet were scrutinized and approved before any batch of diet was released for use. Certificates of analysis were routinely provided by the water supplier.
Certificates of analysis were also received from the suppliers of the softwood based bark-free fiber bedding and Aspen chew blocks.
No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

Route Oral, via the diet.

Treated at Constant dietary concentrations (ppm) for each group.

Control (Group 1) Untreated diet of the same batch with corn oil additive in an amount identical to the high dose group.

Frequency Continuously. Fresh diet was given at a minimum frequency of every four days.

Diet A record of the usage of the diets was maintained on all occasions when food consumption was measured. This was performed using the initial weight of the diet container and an on-line data check on completion of the feeding procedure to ensure that all cages were fed the correct amount of diet. No significant discrepancy was found.

Method of preparation
Test item was melted in a waterbath/oven at a maximum temperature of 43°C. On each occasion of the preparation of the premix the required amount of test item and corn oil (at a ratio of 5:1) were weighed into a suitable container. An amount of plain diet that approximately equaled the weight of test item was added and the mixture stirred together using a spoon. A further amount of plain diet (approximately equal to the weight of this mixture) was added and it was stirred well. This doubling up process was repeated until half of the final weight of the 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 at the pre-set r.p.m. (16) to ensure the test item was homogenously dispersed in the diet.

Aliquots of the premix were then diluted with further quantities of plain diet to produce the required dietary concentrations. Additional corn oil was added to achieve an overall corn oil content of 0.32% in each dietary concentration (with the exception of diets for females in lactation for which the overall corn oil content was 0.16%). Each batch of treated diet was mixed for a further 100 cycles at the pre-set r.p.m. (16) in a Turbula mixer.

For the control diet, an amount of diet was added directly to the corn oil and then prepared as indicated for the premix.

Frequency of preparation Weekly, up to ten days in advance of first use.

Storage of formulation Frozen (-10 to -30°C) until required for feeding.

Test item accounting Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

Details on mating procedure:

Mating Procedure - F0 and F1 Cohort 1B Generation
F0 pairing commenced After ten weeks of treatment.

Cohort 1B F1 pairing commenced Approximately 10 weeks after formal F1 generation commencement.

Male/female ratio 1:1 from within the same treatment groups (sibling pairing was not permitted).

Duration of pairing Up to two weeks.

Daily checks for evidence of mating Ejected copulation plugs in cage tray and sperm in the vaginal smear.

Day 0 of gestation When positive evidence of mating was detected.

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:

Formulation Analysis
Stability and homogeneity Before the commencement of treatment, the suitability of the proposed mixing procedure and the homogeneity and stability of the test item in the carrier diet, was determined as part of Covance Study Number: FW19GH. Those investigations demonstrated stability at dietary concentrations of 1000 to 20000 ppm for up to four days at ambient temperature (15 to 25ºC) and for 24 days following frozen storage (-10 to -30ºC).

Achieved concentration Samples of each formulation prepared for administration in Week 1 of the F0 generation, Week 1 of the F1 generation and the last week of treatment for the F1 generation were analyzed for achieved concentration of the test item.

Duration of treatment / exposure:

Duration of Treatment
F0 animals For ten weeks before pairing until termination after litters were weaned.

F1 animals From weaning@ until termination of respective cohort.

Unselected F1 offspring : Retention of limited tissues and organ weights recorded, no direct treatment, killed on Day 22 of age.
Cohort 1A : General toxicity and pathology of the tissues of the male and female reproductive systems.

Treated from weaning to approximately 13 weeks of age.
Cohort 1B : Treated from weaning throughout pairing, gestation and lactation and up to scheduled termination after weaning of the F2 litters.

Although direct treatment started at weaning (or when the offspring start to consume some treated diet whilst still with the dam), all offspring have potential indirect exposure in utero, through the milk during lactation.

Frequency of treatment:

Continuously. Fresh diet was given at a minimum frequency of every four days.

Details on study schedule:

Selection of Offspring to Form F1 Generation
Selection On Day 18 to 20 of age.

Allocation - formal start of F1 generation Nominally Day 28 of age (direct diet administration commenced on Day 21 of age).

Method
Where possible, two males and two females were selected from each selected litter and were allocated to each of the two cohorts. If more were required, up to three males and three females were selected from each selected litter.

Selected animals were microchipped on Day 18-21 of age and separated from littermates on Day 21 of age. Formal commencement of the F1 generation was on Day 21 of age.

Up to two male and two female F1 offspring per group were retained as spares, to provide potential replacement in the event of any mortalities. These spares had body weights and clinical signs monitored weekly and were terminated after commencement of the F1 generation.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm (nominal)

Remarks:

F0 generation

Doses / concentrations 2

Dose / conc.:

4 000 ppm (nominal)

Remarks:

F0 generation

Doses / concentrations 3

Dose / conc.:

8 000 ppm (nominal)

Remarks:

F0 generation

Doses / concentrations 4

Dose / conc.:

16 000 ppm (nominal)

Remarks:

F0 generation

No. of animals per sex per dose:

F0 generation
25 males, 25 females per dose group

PLEASE REFER TO "ANY OTHER INFORMATION ON MATERIALS AND METHODS" FOR FURTHER DETAILS REGARDING DOSE GROUPS

Control animals:

yes

yes, concurrent no treatment

Details on study design:

Rationale for Dietary Concentration Level Selection
The dietary concentrations selected for investigation in this study (0, 4000, 8000 and 16000 ppm for the F0 animals, and 0, 2000, 4000 and 8000 ppm for F0 females during lactation and for the selected F1 generation animals) were chosen in conjunction with the Sponsor based on the results of a preliminary extended one generation study (Covance Study No. BT84SH) in which similar dietary concentrations were investigated.

In that study, dietary administration of D,L-Menthol at inclusion levels up to and including 16000 ppm (or up to 8000 ppm for F0 females during lactation) were well tolerated. There were no premature deaths or test item-related changes in general clinical condition among F0 or selected F1 generation animals. Dose-dependent reductions in mean food consumption were apparent among F0 animals during Days 1-4 of treatment, which were associated with low mean body weight gain or mean body weight loss. Similarly, in the selected F1 generation, mean food consumption was lower than Control from weaning (Day 21 of age) until termination, most notably during Days 21-23 of age, and associated with slightly lower mean body weight gain in the 16000 ppm group. These reductions in food consumption were considered to have occurred as a consequence of distaste of the prepared diets rather than direct systemic toxicity.

Biochemical analysis of the plasma of F0 and selected F1 generation animals revealed some changes in liver enzyme activities and cholesterol, total protein/albumin and creatinine concentrations however the magnitude of these changes were considered not to be adverse. Increases in liver and/or kidney weights were also observed among F0 adults; these increases were also considered not to be adverse. There were no test item-related macroscopic abnormalities detected.

At 16000 ppm, overall achieved doses for the F0 males, and for the F0 females before pairing were approximately 830-850 mg/kg/day. During gestation at 16000 ppm, F0 females received approximately 1000 mg/kg/day, and during lactation where the high dietary concentration was reduced to 8000 ppm, the overall achieved dose was approximately 1300 mg/kg/day. Selected F1 animals in the 16000 ppm group received approximately 2000-2100 mg/kg/day.

Based on the outcome of the preliminary study, and in order to ensure that the achieved mg/kg/day dose for the F0 females during lactation and for the selected F1 generation animals did not excessively exceed the limit dose (1000 mg/kg/day), the dietary concentrations for these study phases were reduced by 50% (ie. to 2000, 4000 and 8000 ppm) from those given to F0 males and F0 females before pairing and during gestation. Review of the data up to the mid-point of Week 3 of the formal F1 generation showed that the achieved dose was rapidly reducing, and therefore the dietary inclusion levels were increased to 2500, 5000 and 10000 ppm from the start of Week 5 of the formal F1 generation. Further review of the data up to the end of Week 5 of the formal F1 generation showed that the achieved dose had continued to reduce, and therefore the dietary inclusion levels were increased to 4000, 8000 and 16000 ppm from the start of Week 7 of the formal F1 generation.

Examinations

Parental animals: Observations and examinations:

Serial Observations
Clinical Observations - F0 and F1 Generation
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).

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.

During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

Clinical Signs
A detailed physical examination was performed on each animal to monitor general health according to the following schedule:

Physical examination Once each week.

After mating of F0 and F1 Cohort 1B females: Days 0, 5, 12, 18 and 20 after mating and Days 1, 7, 14 and 21 of lactation.

Particular attention was paid to possible signs of neurotoxicity such as convulsions, tremor and abnormalities of gait or behavior.

Mortality - F0 and F1 Generation
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.

A complete necropsy was performed in all cases.

Body Weight - F0 and F1 Generation
The weight of animals was recorded as follows:
F0 males Day that treatment commenced.
Each week.
Before necropsy.
F0 females Day that treatment commenced.
Each week until mating detected.
Days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 after mating.
Days 1, 4, 7, 14, 21 and 28 of post partum.
Before necropsy
F1 selected animals From Day 21 of age, twice weekly.
Before necropsy.
Cohort 1B females: Days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 after mating; Days 1, 4, 7, 14 and 21 and 28 of post partum.
Before necropsy.

Food Consumption - F0 and F1 Generation
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:

F0 males Twice weekly until paired for mating.
F0 females Twice weekly until paired for mating.

Days 0-2, 2-4, 4-6, 6-8, 8-10, 10-12, 12-14, 14-16, 16-18, 18-20 after mating.
Days 1-4, 4-7, 7-11, 11-14, 14-17 and 17-21 of lactation.

F1 selected animals From Day 21 of age, twice weekly.
F1 Cohort 1B females: Days 0-2, 2-4, 4-6, 6-8, 8-10, 10-12,
12-14, 14-16, 16-18 and 18-20 after mating, and Days 1-4, 4-7, 7-11, 11-14, 14-17 and 17-21 of lactation.

Food consumption was not recorded for males and females during the period when paired for mating, but recommenced for males once pairing of all the animals had been completed.

From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each relevant phase.

Mating Procedure - F0 and F1 Cohort 1B Generation
F0 pairing commenced After ten weeks of treatment.
Cohort 1B F1 pairing commenced Approximately 10 weeks after formal F1 generation commencement.
Male/female ratio 1:1 from within the same treatment groups (sibling pairing was not permitted).
Duration of pairing Up to two weeks.
Daily checks for evidence of mating Ejected copulation plugs in cage tray and sperm in the vaginal smear.
Day 0 of gestation When positive evidence of mating was detected.
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.

Parturition Observations and Gestation Length - F0 and F1 Cohort 1B Generation
Duration of gestation Time that elapsed between mating and commencement of parturition.
Parturition observations From Day 20 after mating animals were checked 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 noted.

Thyroid Hormone Analysis - TSH and T4
Blood samples were collected at the following occasions:
Occasion Generation Animals
Termination F0 Adults Ten male and ten female animals per group
F1 Offspring Ten litters per group - pooled litter sample Day 4 of age (T4 only)
F1 Offspring Ten male and ten female animals per group on Day 22 of age (one male and one female from up to 20 litters)
F1 Adults - Cohort 1A Ten male and ten female animals per group

Conditions Adults: Following overnight deprivation of food.

Offspring: No overnight deprivation of food.

Blood sample site Adults: Sublingual vein.
Offspring Day 4 of age: Decapitation.
Offspring Day 22 of age : Retro-orbital sinus

Anaesthetic Adults and offspring on Day 22 of age: Isoflurane.
Offspring Day 4 of age: Not required.

Anticoagulant None.

Tubes Greiner Minicollect - with clot activator.

Blood volume Adults and offspring on Day 22 of age: 1 mL.

Offspring Day 4 of age: Maximum possible.

Treatment of samples Samples were kept at ambient temperature (15 to 25°C) for a minimum of 30 minutes prior to centrifugation.

Centrifugation conditions At 2000g for ten minutes at 4°C.

Number of aliquots Adults and offspring on Day 22 of age: Two per animal

Aliquot 1: 0.2 mL serum for T4
Aliquot 2: residual serum for TSH.

Offspring Day 4 of age: Single aliquot for T4 analysis all available collected.
Final storage conditions Deep frozen (approximately -60°C to -90°C).
Fate of samples Aliquot 1 (T4): dispatched to the Department of Bioanalysis, Covance.
Aliquot 2: dispatched to the Department of Biomarkers, Bioanalysis and Clinical Sciences, Covance.

T4 Performed by the Department of Department of Bioanalysis, Covance
TSH Performed by the Department of Department of Biomarkers, Bioanalysis and Clinical Sciences, Covance.


Hematology, Peripheral Blood - F0 and F1 Cohort 1A Generation
Blood samples were collected after overnight withdrawal of food. Sampling was performed on the morning after overnight collection of urine. These animals were, therefore, also deprived of water overnight but had access to water for a minimum period of one hour prior to the commencement of blood sampling procedures. Samples were collected at the following occasions:
Occasion Generation Animals
Termination F0 Adults Ten male and ten female animals per group
F1 Cohort 1A Ten male and ten female animals per group


Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyzer:
• Hematocrit (Hct)*
• Hemoglobin concentration (Hb)
• Erythrocyte count (RBC)
• Mean cell hemoglobin (MCH)*
• Mean cell hemoglobin concentration (MCHC)*
• Mean cell volume (MCV)
• Red cell distribution width (RDW)
• Total leucocyte count (WBC)
• Differential leucocyte count:
• Neutrophils (N)
• Lymphocytes (L)
• Eosinophils (E)
• Basophils (B)
• Monocytes (M)
• Large unstained cells (LUC)
• Platelet count (Plt)

* Derived values calculated in ClinAxys
Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer. Confirmation or a written description from the blood film was made where appropriate.
Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using a Stago STA Compact Max analyzer and appropriate reagent in respect of:
• Prothrombin time (PT) - using IL PT Fibrinogen reagent.
• Activated partial thromboplastin time (APTT) - using IL APTT reagent.


Blood Chemistry - F0 and F1 Cohort 1A Generation
Blood samples were collected after overnight withdrawal of food. Sampling was performed on the morning after overnight collection of urine. These animals were, therefore, also deprived of water overnight but had access to water for a minimum period of one hour prior to the commencement of blood sampling procedures. Samples were collected at the following occasions:
Occasion Generation Animals
Termination F0 Adults Ten male and ten female animals per group
F1 Cohort 1A Ten male and ten female animals per group


Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche Cobas 6000 Analyzer in respect of:
• Alkaline phosphatase (ALP)
• Alanine aminotransferase (ALT)
• Aspartate aminotransferase (AST)
• Gamma-glutamyl transpeptidase (gGT)
• Total bilirubin (Bili)
• Direct bilirubin (BILD)
• Indirect bilirubin (INDC)
• Urea
• Creatinine (Creat)
• Glucose (Gluc)
• Total cholesterol (Chol)
• Sodium (Na)
• Potassium (K)
• Chloride (Cl)
• Calcium (Ca)
• Inorganic phosphorus (Phos)
• Total protein (Total Prot)
• Albumin (Alb)

Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.

Urinalysis - F0 and F1 Cohort 1A Generation
Urine samples were collected after overnight withdrawal of food and water at the following occasions:
Occasion Generation Animals
Termination F0 Adults Ten male and ten female animals per group
F1 Cohort 1A Ten male and ten female animals per group

The individual samples were examined for the following characteristics:
• Using manual methods:
• Clarity and Color (App) - by visual assessment
• Volume (Vol) - using a measuring cylinder
• pH - using a pH meter
• Specific gravity (SG) - by direct refractometry using a SG meter

• Using Multistix reagent strips interpreted using the Clinitek®500 instrument:
• Glucose (Gluc)
• Ketones (Keto)
• Bile pigments (Bili)
• Blood pigments (UBld)

• Using a Cobas 6000 Analyzer:
• Protein - total (T-Prot) and concentration (Prot)
• Sodium - total (T-Na) and concentration (U-Na)
• Potassium - total (T-K) and concentration (U-K)
• Chloride - total (T-Cl) and concentration (U-Cl)

A microscopic examination of the urine sediment was performed. An aliquot of the urine sample was centrifuged, stained with Kova stain and the resulting deposit spread on a microscope slide. The number of elements seen in nine high or low power fields (HPF or LPF) was recorded in the raw data and entered onto the database and the number seen /HPF or /LPF was derived from these data as described below.
• Epithelial cells (Epi)
• Leucocytes (WBC)
• Erythrocytes (RBC)
• Casts
• Other abnormal components (A)

The slide was also examined for abnormalities in spermatozoa and crystals.

Oestrous cyclicity (parental animals):

Estrous Cycle Monitoring - F0 and F1 Cohort 1B Generation
Dry and wet smears were taken as follows:
Dry smears - F0 females only For 15 days before pairing, using cotton swabs.
Wet smears - F0 and F1 Cohort 1B females After pairing until evidence of mating confirmed.

For four days before scheduled termination (nominally Days 25 to 28 post partum). Females that failed to litter or mate were retained and smeared for four days starting on the day on which the first batch of ‘true’ Day 25 post partum females started smearing, and were then killed with that first batch of females.

Sperm parameters (parental animals):

Sperm Analysis - F0 and F1 Cohort 1A Generation
Immediately after scheduled sacrifice of each F0 and F1 Cohort 1A male, the left vas deferens, epididymis and testis were removed and the epididymis and testis were weighed.

The following tests were performed:
F0 generation
Sperm motility: all groups A sample of sperm was expressed from the left vas deferens (right tissues used for Group 1M 22 and 24) into prewarmed (target 37°C) medium M199, which contained 0.5% w/v bovine serum albumin (BSA Fraction V). A sample for assessment was taken into a 100 µm depth cannula by capillary action and, at least 200 sperm per animal analyzed using the Hamilton Thorne IVOS II Computer Assisted Sperm Analyser (CASA) (Group 1M 19 unable to assess 200).

Sperm morphology: Groups 1 and 4 A 200 L aliquot of the sperm/medium mixture (described above) was diluted with 800 L of 10% neutral buffered formalin. After staining with nigrosine and eosin an air-dried smear was prepared. Slides were examined by light microscopy for the assessment of sperm morphology. At least 200 sperm were assessed for each male, where possible (Group 1M 19 and Group 4M 83 and 86 unable to assess 200).

Sperm morphology: Groups 2 and 3 Fixed samples retained for possible future assessment.

Sperm count: Groups 1 and 4 The left cauda epididymis of each male (right tissues used for Group 1M 22 and 24) was weighed and then the tunica was removed, then homogenized for at least 30 seconds in 10 mL of a mixture of 0.9% saline and 0.01% merthiolate (SM). An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for sperm count using CASA.

Sperm count: Groups 2 and 3 Samples frozen for possible future assessment.

Homogenization-resistant spermatid counts: Groups 1 and 4 After removal of the tunica, the left testis of each male (right tissues used for Group 1M 22 and 24) then homogenized for at least 30 seconds in 25 mL of SM. An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for homogenization resistant spermatid count using CASA.

Homogenization-resistant spermatid counts: Groups 2 and 3 Samples frozen for possible future assessment.

Cohort 1A F1 generation
Sperm motility: all groups A sample of sperm was expressed from the left vas deferens into prewarmed (target 37°C) medium M199, which contained 0.5% w/v bovine serum albumin (BSA Fraction V). A sample for assessment was taken into a 100 µm depth cannula by capillary action and, at least 200 sperm per animal analysed using the Hamilton Thorne IVOS II Computer Assisted Sperm Analyzer (CASA).

Sperm morphology: Groups 1 and 4 A 200 L aliquot of the sperm/medium mixture (described above) was diluted with 800 L of 10% neutral buffered formalin. After staining with nigrosine and eosin an air-dried smear was prepared. Slides were examined by light microscopy for the assessment of sperm morphology. At least 200 sperm were assessed for each male.
Sperm morphology: Groups 2 and 3 Fixed samples retained for possible future assessment.
Sperm count: Groups 1 and 4 The left cauda epididymis of each male was weighed and then the tunica was removed, then homogenized for at least 30 seconds in 10 mL of a mixture of 0.9% saline and 0.01% merthiolate (SM). An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for sperm count using CASA. (Group 4M 469, whole cauda weight was not recorded in error, cauda portion weight used to derive data)

Sperm count: Groups 2 and 3 Samples frozen for possible future assessment.

Homogenization-resistant spermatid counts: Groups 1 and 4 After removal of the tunica, the left testis of each male then homogenized for at least 30 seconds in 25 mL of SM. An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for homogenization resistant spermatid count using CASA.

Homogenization-resistant spermatid counts: Groups 2 and 3 Samples frozen for possible future assessment.

Litter observations:

Records Made During Littering Phase - F0 and F1 Cohort 1B Generation
The records maintained were as follows:
Clinical observations Observed approximately 24 hours after birth (Day 1 of age) and then daily for evidence of ill-health or reaction to treatment.
On Day 1 of age, all offspring received a qualitative assessment of body temperature, state of activity and reaction to handling.

Litter size Daily records were maintained of mortality and consequent changes in litter size from Days 1-21 of age.
On Day 4 of age, litters containing more than ten offspring were reduced to ten by random culling, leaving, whenever possible, five male and five female offspring in each litter.
All culled offspring were macroscopically examined, with thyroid hormone samples collected from 10 x F1 litters per group.

Sex ratio of each litter Recorded on Days 1, 4 (before and after culling) and on Day 21 of age.

Individual offspring body weights Recorded on Days 1, 4 (before culling), 7, 14 and 21 of age. F2 off spring were also weight on Day 22 of age.

Weaning of offspring The dam was removed from the litter cage and offspring were weaned on Day 21 of age (F1) and Day 22 of age (F2).

Ano-genital distance Day 1 of age - all offspring.

Nipple/areolae count Day 13 of age - male offspring. (F1 and F2)

Day 21 of age - male offspring (F2)

Postmortem examinations (parental animals):

Time of Necropsy
F0 males After at least 18 weeks of treatment and after weaning of the F1 animals, after confirmation that no further mating was required.
F0 and Cohort 1B females Day 28 post partum.
F0 and Cohort 1B females failing to produce a viable litter Terminated with first cohort of females with live litters.
Unselected F1 and F2 offspring Day 4 and Day 22 of age.
Cohort 1A animals At approximately 13 weeks of age.
Cohort 1B males After weaning of the F2 offspring

All animals, including surplus offspring culled on Day 4 of age and unselected offspring on Day 22 of age were subject to a complete macroscopic examination. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative. Decedents offspring 21 days of age, (found dead or welfare kill), where possible, were examined and carcass retained.

For F0 and F1B Females, the number of implantation sites was counted.

For females of Cohort 1A, counts were performed for the number of ovarian follicles (primordial and small growing) and corpora lutea.


PLEASE REFER TO ATTACHED TABLE RE "PATHOLOGY PROCEDURES - F0 (PARENTAL) ANIMALS

Postmortem examinations (offspring):

PLEASE REFER TO ATTACHED TABLES:
* Pathology Procedures - Cohort 1A - Tissue retention, organ weights and tissue processing
* Pathology Procedures - Cohort 1B - Tissue rentention, organ wights and tissue processing

Statistics:

Data Evaluation
This report contains serial observations pertaining to all weeks of treatment completed, together with signs data collected during the necropsy period. No serial observations relating to the acclimatization period are included in this report.

Summary statistics (e.g. means and standard deviations) presented in this report were calculated from computer-stored individual raw data. The summary statistics and the individual data were stored in the computer to a certain number of decimal places, different for each parameter. For presentation purposes, however, they were usually rounded to fewer places. It is, therefore, not generally possible to reproduce the presented means and standard deviations exactly using the presented individual data.

Throughout the report the following abbreviations were used:
M Male
F Female
SD Standard deviation
N Number contributing to the mean (normally the number of animals/litters)

Reproductive indices:

Mating Performance and Fertility - F0 and F1 Cohort 1B Generation
Individual data was tabulated. Group values were calculated for males and females separately for the following:
Percentage mating (%) = (Number of animals mating / Animals paired) x 100

Conception rate (%) = (Number of animals achieving pregnancy/ Animals mated) x 100

Fertility index (%) = (Number of animals achieving pregnancy / animals paired) x 100

Gestation Length and Gestation Index - F0 and F1 Cohort 1B Generation
Gestation length was 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.

Gestation index was calculated for each group as:

Gestation index (%) = (Number of live litters born/ Animals paired) x 100

Offspring viability indices:

Survival Indices - F0 and F1 Cohort 1B Generation
The following were calculated for each litter:

Post implantation survival index (%) = (Total number of offspring born / Total number of uterine implantation sites) x 100

Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.

Live birth index (%) = (Number of offspring on Day 1 after littering / Total number of offspring born) x 100

Viability index (%) = (Number of live offspring on Day 4 before culling / Number of live offpsring on Day 1 after littering) x 100

Lactation index (%) = (Number of live offspring on Day 21 after littering / Number of live offspring on Day 4 (after culling)) x 100

Group mean values were calculated from individual litter values.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No test item-related changes in general clinical condition were observed during the routine physical examination procedures for F0 generation animals throughout the study.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were two premature deaths among F0 generation animals.

One Group 3 female given 8000/4000 ppm (No. 264) was killed for reasons of animal welfare on Day 1 of lactation due to a prolapsed uterus. This female had previously given birth to 13 live pups. Macroscopic examination revealed dark contents in the duodenum, ileum and jejunum (considered likely to represent maternal consumption of the placentae etc), inactive/pale mammary tissue and small spleen; there were no notable histopathological changes.

One Group 2 female given 4000 ppm (No. 241) was found dead on Day 44 after mating (i.e. non-pregnant). Macroscopic examination revealed the uterus was distended, fluid was present in the abdomen and some lymph nodes were enlarged. Histopathological examination revealed this animal to have a notable pyometra and this was considered to be the cause of death. Other histopathological changes, including, increased hematopoiesis and increased cellularity in lymph nodes were associated with this.

These premature deaths were considered unrelated to treatment with D,L-Menthol.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Following the commencement of treatment, when compared to Controls males given 8000 or 16000 ppm showed a slight, but statistically significant, dose-dependent reduction in mean body weight gain during Week 1. Thereafter, mean body weight gain during Weeks 2-4 was essentially similar to Controls, however further reductions in mean weight gain were apparent during Weeks 5, 6, 8 and 10, with low mean body weight gain also evident at 16000 ppm in Weeks 14 to 18, and at 8000 ppm in Weeks 16 and 17. At 4000 ppm, mean body weight gain was slightly low during Week 6, 10, 16 and 17. Overall mean body weight gain during Weeks 0-18 was 7% and 11% lower than Control at 8000 and 16000 ppm, respectively; overall mean body weight gain at 4000 ppm during Weeks 0-18 was unaffected.

Females given 16000 ppm showed slightly, but statistically significantly, low mean body weight gain when compared to Controls during Week 1 of treatment; thereafter, mean body weight gain during Weeks 2-10 of the pre-pairing period was essentially similar to Control, with overall mean body weight gain during Weeks 0-10 being 7% lower than Control. Mean body weight gain was unaffected by treatment for females given 4000 or 8000 ppm during the 10-week pre-pairing period.

After mating, females given 16000 ppm showed low overall mean body weight gain during Days 0-20 of gestation (17% lower than Control, and attaining statistical significance; contributed to by the lower mean litter size recorded in this group). The body weight performance of females given 4000 or 8000 ppm during gestation was unaffected.

Following parturition, when the dietary concentrations were reduced by 50% in all groups, the overall mean body weight gain of females given 8000 ppm during Days 1-21 of lactation was statistically significantly higher than Control, with the pattern of weight gain during Days 1-4 and 14-21 of lactation being different to Controls. These females also showed a higher level of body weight loss during Days 21-28 of lactation, following weaning of the offspring. The body weight performance of females given 2000 or 4000 ppm during lactation was considered to be unaffected by treatment.

PLEASE REFER TO THE FOLLOWING ATTACHED TABLES
Table 11.7 Body weight and body weight change_F0 males
Table 11.8 Body weight and body weight change_Females during gestatuin_F0
Table 11.9 Body weight and body weight change_Lactating females_F0

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 16000 ppm, group mean food consumption during Weeks 1-10 (Days 1-71) of the pre-pairing treatment period was consistently lower than Control for males and females, particularly during Day 1-4 (Week 1). With the exception of the first three days of treatment, the majority of differences from Control were small but most attained statistical significance. A similar pattern continued for males after pairing (Weeks 12 to 18; Days 78-130).

At 8000 ppm, group mean food consumption was statistically significantly lower than Control during Days 1-4. Thereafter, in males mean food intake at this dietary inclusion level was variable to the end of Week 10 of the pre-pairing period and in Weeks 12-18 (after pairing) but with a tendency towards being slightly lower than Control, with occasional recording periods attaining statistical significance. In females, mean food consumption was statistically significantly lower than Control throughout Weeks 3-10 of the pre-pairing treatment period (Day 15-71).

At 4000 ppm, the mean food intake of females during Days 1-4 was marginally, but statistically significantly, lower than Control. Food consumption was also statistically significantly lower than Control for the majority of the recording periods during Weeks 3-10 (Days 18-71). Food consumption during Weeks 1-18 for males given 4000 ppm was not clearly affected, although a trend towards slightly lower mean food consumption than Controls was observed during Weeks 6-10, Weeks 13-15 and Week 18 (Days 36-71, 85-92 and 123-130).

After mating, group mean food consumption was statistically significantly lower than Control for females during Days 0-10 and Days 12-14 of gestation at 8000 ppm, and throughout the majority of the gestation period at 16000 ppm, with a dose-dependent trend apparent; the mean food intake of females given 4000 ppm during gestation was not clearly affected.

Following parturition, when the dietary concentrations were reduced by 50% in all groups, mean food consumption was statistically significantly lower than Controls among females given 8000 ppm from Day 4 to Day 14 of lactation. From Day 14-21 of lactation at 8000 ppm and from Day 17-21 of lactation at 4000 ppm, mean food consumption was also statistically significantly lower than Control, however it cannot be ascertained whether this was reflective of slightly lower food intake by the offspring as they approached weaning, or by the parent females, or both.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Analysis of hematological parameters at scheduled termination of the F0 animals did not reveal any changes which were clearly attributable to D,L-Menthol administration.

All differences from control were minor, limited to one sex and/or lacked a dose response relationship, and were therefore attributed to normal biological variation; these included the slight increase in red blood cell counts in males given 16000 ppm, slightly low mean cell hemoglobin concentrations in males given 8000 or 16000 ppm, slight decrease in total white blood cell and lymphocyte concentrations in males given 16000 ppm, slightly low monocyte concentrations in males given 8000 or 16000 ppm and in all groups of treated females, slightly low basophil and large unstained cell concentrations in all groups of treated males, and slightly shortened prothrombin time in males given 16000 ppm.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Biochemical analysis of the plasma at scheduled termination of the F0 animals revealed when compared to Controls, statistically significantly slightly increased mean gamma glutamyl transpeptidase concentrations in males given 16000 ppm and females given 16000/8000 ppm. Urea concentrations were increased in males given 16000 ppm, a dose-dependent decrease in creatinine concentrations was evident in females given 16000/8000 or 8000/4000ppm, and glucose concentrations were low (although in the absence of a dose response relationship) in all groups of treated males and in females given 16000/8000 or 8000/4000ppm.

In all groups of treated females, changes in electrolyte concentrations were apparent, with slight increases in sodium and calcium concentrations when compared to Controls; mean phosphorus concentrations were also slightly increased in females given 8000/4000 or 4000/2000 ppm although phosphorus concentrations in females given 16000/8000 ppm were unaffected.

When compared to Controls, albumin/globulin ratio was slightly low in males given 16000 ppm and females given 16000/8000 ppm due to a marginal increase in total protein concentrations.

All other biochemical differences from Controls observed at scheduled termination were minor, limited to one sex and/or lacked a clear dose relationship, and were therefore attributed to normal biological variation; this included the slight decrease in alkaline phosphatase activity in females given 16000/8000 ppm and the low chloride concentrations in males given 16000 ppm.

PLEASE REFER TO THE FOLLOWING ATTACHED TABLE
Table 11.19 Blood cheistry at termination_F0

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Analysis of the clarity/colour and composition of the urine and microscopy of the urine sediment of F0 animals prior to scheduled termination did not reveal any test item-related changes.

Males given 16000 ppm showed slightly low urinary potassium concentration when compared to Controls; total potassium concentration was unaffected and therefore the difference in urinary concentrations was attributable to the slightly higher urine volume output in this group of males, and no toxicological significance was inferred.

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):

Treatment Related Findings
Changes related to treatment with D,L-Menthol were seen in the kidneys of males and the liver of females.

Kidneys
In males, hyaline droplet accumulation was seen with associated changes; multifocal tubular basophilia and/or cast formation was present in most animals given 8000 or 16000 ppm and some given 4000 ppm. Tubular dilation, diffuse or multifocal, was also present in some males given 16000 ppm and one given 8000 ppm.
 
Summary of treatment related findings in the kidneys for F0 males killed after completion of treatment
Group/sex 1M 2M 3M 4M
Dietary level (ppm) 0 4000 8000 16000

Accumulation, Hyaline Droplets
Minimal 0 7 13 3
Slight 0 2 10 13
Moderate 0 0 0 7
Total 0 9 23 23

Basophilia, Tubular, Multifocal
Minimal 0 1 15 10
Slight 0 2 4 7
Total 0 3 19 17

Cast(s)
Minimal 1 2 9 9
Slight 0 0 0 1
Total 1 2 9 10

Dilation, Tubular, Diffuse
Minimal 0 0 0 4
Slight 0 0 0 6
Total 0 0 0 10

Dilation, Tubular, Multifocal
Minimal 0 0 1 2
Total 0 0 1 2

Number of tissues examined 25 25 25 25

Liver
A dose dependent increase in centrilobular hypertrophy was present in the liver of females given D,L-Menthol at all dietary levels.

Summary of treatment related findings in the liver for F0 females killed after completion of treatment

Group/sex 1F 2F 3F 4F
Dietary level (ppm) 0 4000/ 8000/ 16,000
2000 4000 8,000
Hypertrophy, Centrilobular
Minimal 0 3 10 16
Total 0 3 10 16

Number of tissues examined 25 24 24 25

Incidental Findings
A fibroadenoma was present in the mammary gland of Group 2 female 239 given D,L Menthol at 4000/2000 ppm. This was considered unrelated to treatment in view of its isolated incidence in the low dose group, and the fact that these tumours are sometimes seen as background findings in female rats of this age and strain.

Congestion in the stomach seen occasionally in treated females was considered incidental due to the low incidence and occurrence in only one sex. Focal/multifocal mineralization in the stomach of females was seen in both control and high dose animals, was of low severity and is not considered to be related to the administration of D,L-Menthol.

Seminiferous tubules of the testes were evaluated with respect to their stage in the spermatogenic cycle and the integrity of various cell types present within the different stages. No cell or stage specific abnormalities were noted. The qualitative examination of the ovary and staging of the estrus cycle revealed no abnormality.

All other findings were considered to be incidental and unrelated to treatment.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid Hormone Analysis - TSH and T4
Mean serum T4 concentrations in F0 and F1 Cohort 1A adult animals and in Day 22 offspring given D,L-Menthol were unaffected by treatment at all dose levels investigated, with concentrations essentially similar to endogenous levels observed in the control matrix used to prepare the QC samples. It was noted that T4 concentrations for F1 female offspring in the high dose group were statistically significantly higher than Control, however in the absence of a similar difference in male offspring on Day 22 of age, this difference was considered incidental and unrelated to D,L-Menthol.

Mean serum TSH concentrations in F0 and F1 Cohort 1A adult animals and in Day 22 offspring given D,L-Menthol were unaffected by treatment at all dose levels investigated. F0 females given 16000/8000 ppm showed a statistically significant increase in TSH concentrations when compared to Controls. A similar difference was not apparent in the F0 males or in the adult F1 Cohort 1A animals, and the results for the F0 females in the high dose group showed a relatively high degree of intra-animal variability, therefore this difference was considered incidental and unrelated o D,L-Menthol.

Incidental Findings
A fibroadenoma was present in the mammary gland of Group 2 female 239 given D,L Menthol at 4000/2000 ppm. This was considered unrelated to treatment in view of its isolated incidence in the low dose group, and the fact that these tumours are sometimes seen as background findings in female rats of this age and strain.

Congestion in the stomach seen occasionally in treated females was considered incidental due to the low incidence and occurrence in only one sex. Focal/multifocal mineralization in the stomach of females was seen in both control and high dose animals, was of low severity and is not considered to be related to the administration of D,L-Menthol.

All other findings were considered to be incidental and unrelated to treatment.

Seminiferous tubules of the testes were evaluated with respect to their stage in the spermatogenic cycle and the integrity of various cell types present within the different stages. No cell or stage specific abnormalities were noted. The qualitative examination of the ovary and staging of the estrus cycle revealed no abnormality.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycle regularity was assessed for the F0 females for 15 days prior to pairing. There was no effect on the number of females showing regular estrous cycles at any dietary inclusion level investigated. It was noted that females given 16000 ppm showed a slightly different distribution of 4-day or 5-day cycles when compared with Controls, however this was considered incidental and unrelated to D,L-Menthol administration.

Estrous cycles at termination were unaffected by treatment at all dietary concentrations.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

At scheduled termination, the motility and motion of the sperm, testicular and cauda epididymal sperm counts and sperm morphology were unaffected by treatment with D,L Menthol at all dietary concentrations investigated. It was noted that the spermatid count (millions.g) in the testis for males given 16000 ppm was slightly lower than Control, with statistical significance attained, however in the absence of any effect upon sperm concentrations in the epididymis, this is considered to be of no biological significance and unrelated to treatment with D,L-Menthol.

PLEASE REFER TO THE FOLLOWING ATTACHED TABLE
Table 11-28_Sperm analysis_F0

Reproductive performance:

no effects observed

Description (incidence and severity):

There was no effect of D,L-Menthol administration on pre-coital interval, with all mating pairs showing positive mating evidence at the first estrus opportunity. Mating performance and fertility, and gestation length and gestation index were unaffected at all dietary concentrations investigated.

There were no clinical signs observed among the F1 offspring that were considered to be related to parental treatment with D,L-Menthol.

In the 16000/8000 ppm group, the mean number of uterine implantation sites was statistically significantly lower than Control. As a consequence, the total offspring born and mean number of live offspring on Days 1 and 4 of age (before litter standardisation) was also lower than Control. There was no effect of D,L-Menthol on mean litter size at 8000/4000 or 4000/2000 ppm.

Offspring survival indices to Day 21 of age and sex ratio were unaffected by D,L-Menthol administration at all dietary concentrations investigated.

The birth weight of male and female F1 offspring was essentially similar in all groups.

In the 16000/8000 ppm group, group mean body weight gain during Days 1-4 of age was statistically significantly higher than Control in male and female offspring. Thereafter, from Day 4 of age, mean body weight gain among these offspring was statistically significantly lower than Control, such that group mean absolute body weight on Day 21 of age was 17% lower than Control in male and female offspring.

At 8000/4000 ppm, offspring body weight gain was unaffected during Days 1-7 of age, however during Days 7-21 of age mean body weight gain was lower than Control, with statistical significance attained for the differences except for Day 7-14 of age in males. Consequently, mean absolute body weights on Day 21 of age were 6% lower than Control in both sexes.

Offspring body weight gain at 4000/2000 ppm was unaffected by treatment.
At scheduled termination on Day 22 of age, the group mean terminal body weight of male and female offspring in the 16000/8000 ppm group and of female offspring in the 8000/4000 ppm group were lower than Control, reflecting the lower body weight gain observed prior to weaning.

Group mean absolute spleen, thymus and uterus weights and body weight relative spleen weights were markedly lower than Controls in both sexes at 16000/8000 ppm and in females at 8000/4000 ppm. Body weight relative brain weights were higher than Control in both sexes at 16000/8000ppm.

There were no test item-related macroscopic abnormalities detected among F1 offspring killed or dying prematurely, among F1 offspring culled on Day 4 of age or among the unselected F1 offspring at scheduled termination on Day 22 of age.

PLEASE REFER TO THE FOLLOWING ATTACHED TABLES
Table 11-21_Litter Size_F1
Table 1162_Litter size_F2

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

>= 419 - <= 499 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

not determinable

2

Key result

Dose descriptor:

NOAEL

Effect level:

>= 455 - <= 594 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

3

Key result

Dose descriptor:

NOAEL

Effect level:

8 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: reduced litter size at birth up to day 4 pp for F1 and F2 offspring

4

Key result

Dose descriptor:

LOEL

Effect level:

8 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

Remarks on result:

other:

Remarks:

increased gamma glutamyl transpeptidase

5

Key result

Dose descriptor:

NOAEL

Effect level:

4 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Target system / organ toxicity (P0)

1

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

8 000 ppm

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

no

2

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

8 000 ppm

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Results: P1 (second parental generation)

Effect levels (P1)

Remarks on result:

not determinable

Target system / organ toxicity (P1)

Critical effects observed:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No test item-related changes in general clinical condition were observed during the routine physical examination procedures for F1 generation Cohort 1A or Cohort 1B animals throughout the study.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

One premature death in Cohort 1A occurred between weaning on Day 21 of age and formal commencement of the F1 generation (nominal Week 4 of age). Male number 436, receiving 2000 ppm, was found dead on Day 27 of age. There were no ante mortem signs observed, and no macroscopic abnormalities were detected. Histopathological examination revealed agenesis in the testes, a small scar in the kidneys and apoptosis in the thymus, with no obvious histopathological cause of death identified. In the absence of similar incidences at this or higher dietary inclusion levels, this isolated premature death was considered incidental and unrelated to D,L-Menthol. This animal was replaced at the start of the formal F1 generation (at nominal Week 4 of age) to ensure a full complement of Cohort 1A animals were available for assessment.

One premature death in Cohort 1B occurred after completion of pairing. Male number 540, receiving 4000/5000/8000 ppm, was found dead in Week 12 of the formal F1 generation. There were no ante mortem signs observed and body weight performance had been in line with expectation. Macroscopic abnormalities comprised distension of the caecum, colon and rectum; these findings may be indicative of the fact that the animal had been dead for a few hours and gaseous build-up had occurred in the lower GI tract. This premature death was considered unrelated to D,L-Menthol administration.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The group mean absolute body weight of selected F1 males and females on Day 21 of age in the 4000/5000/8000 or 8000/10000/16000 ppm groups was statistically significantly lower than Control, reflecting the lower mean body weight gain recorded during the pre-weaning period.

In the 8000/10000/16000 ppm group, selected F1 males and females showed statistically significantly low mean body weight gain during Days 21-24 of age, and this reduced weight gain persisted for males during Days 1-8 of the formal F1 generation (which commenced at nominal Week 4 of age). From the second week of the formal F1 generation onwards, the body weight performance of males given 8000/10000/16000 ppm was variable, with a tendency towards being lower than Control, such that overall mean body weight gain during Days 21 of age to Day 61 of the formal F1 generation (prior to termination of the Cohort 1A animals) was 8% lower than Controls. Overall body weight gain for males from Day 21 of age to Day 131 of the formal F1 generation (termination of Cohort 1B animals) was 9% lower than Control. The body weight gain of females given 8000/10000/16000 ppm from Day 1-61 of the formal F1 generation was not clearly affected by treatment, with overall mean body weight gain from Day 21 of age to Day 61 of the formal F1 generation being 6% lower than Control.

In the 4000/5000/8000 ppm group, the mean body weight gain of males was generally slightly lower than Control, with differences in occasional recording periods attaining statistical significance. Overall mean body weight gain from Day 21 of age to Day 61 of the formal F1 generation (prior to termination of the Cohort 1A animals) or to Day 131 of the formal F1 generation (termination of the Cohort 1B animals) was 4% lower than Controls. The body weight performance of females given 4000/5000/8000 ppm was unaffected by D,L Menthol.

The mean body weight performance of selected F1 generation males and females given 2000/2500/4000 ppm was unaffected by treatment.

When compared to Controls, Cohort 1B females given 8000/10000/16000 ppm showed slightly variable body weight performance during Days 0-18 of the gestation period and notably low mean body weight gain during Days 18-20 of gestation, the primary fetal growth period. Overall mean body weight gain during Days 0-20 of gestation was 14% lower than Control (contributed to by a slightly low mean litter size in this group); mean body weight gain for females given 2000/2500/4000 ppm or 4000/5000/8000 ppm was considered unaffected during gestation.

Following parturition, when the dietary concentrations were reduced by 50% in all groups, the overall mean body weight gain of females given 8000 ppm during Days 1-21 of lactation was statistically significantly higher than Control, with the pattern of weight gain during Days 1-7 and 14-21 of lactation being different to Controls. These females also showed a higher level of body weight loss during Days 21-28 of lactation, following weaning of the offspring. The body weight performance of females given 2000 or 4000 ppm during lactation was considered to be unaffected by treatment.

Offspring Body Weight
The birth weight of male and female F1 offspring was essentially similar in all groups.

In the 16000/8000 ppm group, group mean body weight gain during Days 1-4 of age was statistically significantly higher than Control in male and female offspring. Thereafter, from Day 4 of age, mean body weight gain among these offspring was statistically significantly lower than Control, such that group mean absolute body weight on Day 21 of age was 17% lower than Control in male and female offspring.

At 8000/4000 ppm, offspring body weight gain was unaffected during Days 1-7 of age, however during Days 7-21 of age mean body weight gain was lower than Control, with statistical significance attained for the differences except for Day 7-14 of age in males. Consequently, mean absolute body weights on Day 21 of age were 6% lower than Control in both sexes.

Offspring body weight gain at 4000/2000 ppm was unaffected by treatment.

PLEASE REFER TO THE FOLLOWING ATTACHED TABLES
Table 11.25 Body weight and body weight change_F1
Table 11.45 Body weight and body weight change_Males abd Cohort 1A and 1B females
Table 11.66 Body weight and body weight change_Offspring F2

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Group mean food consumption for males and females in the 8000/10000/16000 ppm group was consistently lower than Control from Day 21 of age to Week 19 of the formal F1 generation (termination of the Cohort 1B animals).

At 4000/5000/8000 ppm, the mean food intake of males and females was lower than Control during Days 21-27 of age; in females, this difference continued throughout the formal F1 generation up to Week 10 (pairing of the Cohort 1B animals), and for males from Week 5 to Week 10 of the formal F1 generation, but was unaffected for Cohort 1B males from Week 12 to Week 19 of the formal F1 generation.

For males given 2000/2500/4000 ppm, mean food consumption during the formal F1 generation was slightly variable, but considered unaffected by D,L-Menthol administration. The mean food intake of females in this group was slightly lower than Control throughout the majority of the formal F1 generation up to pairing for the Cohort 1B females.

After mating, group mean food consumption was statistically significantly lower than Control for females during Days 0-2 and Days 4-14 of gestation at 8000 ppm, and throughout the majority of the gestation period at 16000 ppm, generally with a dose-dependent trend apparent; the mean food intake of females given 4000 ppm during gestation was not clearly affected.

Following parturition, when the dietary concentrations were reduced by 50% in all groups, mean food consumption was statistically significantly lower than Controls among females given 8000 ppm from Day 4 to Day 14 of lactation. From Day 14-21 of lactation at 8000 ppm and from Day 14-17 of lactation at 4000 ppm, mean food consumption was also statistically significantly lower than Control, however it cannot be ascertained whether this was reflective of slightly lower food intake by the offspring as they approached weaning, or by the parent females, or both.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Analysis of hematological parameters at scheduled termination of the Cohort 1A animals did not reveal any changes which were clearly attributable to D,L-Menthol administration.

All differences from control were minor, limited to one sex and/or lacked a dose response relationship, and were therefore attributed to normal biological variation; these included the slight decrease in haematocrit and red blood cell counts in females given 8000/10000/16000 ppm, and the slight non dose-dependent increase in mean cell haemoglobin and mean cell volume in all groups of treated males.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Cohort 1A
Biochemical analysis of the plasma at scheduled termination of the Cohort 1A animals revealed when compared to Controls, statistically significantly slightly increased mean gamma glutamyl transpeptidase concentrations in females given 4000/5000/8000 ppm and in both sexes given 8000/10000/16000 ppm. Urea concentrations were increased in males given 4000/5000/8000 ppm or 8000/10000/16000 ppm, a non dose-dependent decrease in creatinine concentrations was evident in all groups of treated females, with statistical significance attained at 8000/10000/16000 ppm, and cholesterol concentrations were slightly increased in both sexes given 8000/10000/16000 ppm.

Males given 4000/5000/8000 ppm or 8000/10000/16000 ppm showed an increase in calcium concentrations when compared to Controls, although in the absence of a dose response relationship.

When compared to Controls, albumin/globulin ratio was slightly low in all groups of treated females due to a marginal non dose-dependent increase in total protein concentrations. Males given 8000/10000/16000 ppm showed a slight increase in total protein and albumin concentrations, although albumin/globulin ratio was unaffected.

All other biochemical differences from Controls observed at scheduled termination were minor, limited to one sex and/or lacked a clear dose relationship, and were therefore attributed to normal biological variation.

PLEASE REFER TO THE FOLLOWING ATTACHED TABLE
Table 11.60 Blood chemistry_Termination_Cohort 1A_F1

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Cohort 1A
When compared to Control, test item-related urinary changes observed in the Cohort 1A animals were limited to a slight increase in acidity of the urine of males in all treated groups.

Males given 8000/10000/16000 ppm showed slightly low urinary potassium and chloride concentration when compared to Controls; total potassium and chloride concentrations were unaffected and therefore the difference in urinary concentrations were attributable to the slightly higher urine volume output in this group of males, and no toxicological significance was inferred.

Sexual maturation:

effects observed, treatment-related

Description (incidence and severity):

Among selected F1 males given 8000/10000/16000 ppm group, when compared to Controls a statistically significant marked 3.2-day delay in attainment of balano preputial separation was apparent, in the absence of an associated effect on absolute body weight on the day of attainment; balano preputial separation was, however, achieved in all F1 males when comparable body weights were attained indicating that this observation may be secondary to reductions in body weight gain. There was no effect of treatment on the age or body weight at attainment among males given 2000/2500/4000 or 4000/5000/8000 ppm.

Among selected F1 generation females, there was no clear effect on the age or body weight at attainment of vaginal opening at any dietary inclusion level investigated. It was noted that the mean age of attainment for females in the 8000/10000/16000 ppm group was 1.3-days older than Controls, with statistical significance attained. Review of the individual data revealed only 4/40 females which attained vaginal opening after Day 37 of age (the age of the oldest concurrent Control to attain this developmental milestone), and since the animals are only assessed once per day (such that the accuracy of recording is 1-day) this slight increase in the age of attainment of vaginal opening in the 8000/10000/16000 ppm group was considered fortuitous and unrelated to D,L-Menthol.
Cohort 1A females were assessed daily from the day of vaginal opening until the first estrus smear was observed. There was no clear effect of treatment on the interval between vaginal opening and first estrus, although it was noted that one female given 4000/5000/8000 ppm and two females given 8000/10000/16000 ppm achieved first estrus slightly later than the latest Control female.

PLEASE REFER TO THE FOLLOWING ATTACHED TABLE
Table 11.51 Sexual maturation_Age and body weight_Cohort females

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

There was no evidence of an effect of parental D,L-Menthol administration on the ano-genital distance of the F1 offspring on Day 1 of age.

In all treated groups, adjusted mean ano-genital distance was statistically significantly decreased when compared to Controls. Differences were, however, very small (0.1-0.2mm), and there was no dose response relationship apparent, therefore these minor differences were considered to be incidental and unrelated to D,L-Menthol.

Nipple retention in male pups:

effects observed, non-treatment-related

Description (incidence and severity):

All F1 male offspring were examined on Day 13 of age for the presence of nipples. There were no cases of nipples (areolae) being apparent in male offspring in the Control or 4000/2000 ppm litters, however in the 8000/4000 ppm group 13 male offspring (in a total of 6 litters) were found to have nipples (areolae) and 48 male offspring in 16000/8000 ppm group (in a total of 18 litters) were found to have nipples (areolae).

During Week 5 of the formal F1 generation, all selected F1 males which were recorded as having nipples (areolae) apparent on Day 13 of age were reassessed; the nipples (areolae) were no longer apparent indicating that long-term nipple retention had not occurred.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Off spring Organ Weights
At scheduled termination on Day 22 of age, the group mean terminal body weight of male and female offspring in the 16000/8000 ppm group and of female offspring in the 8000/4000 ppm group were lower than Control, reflecting the lower body weight gain observed prior to weaning.

Group mean absolute spleen, thymus and uterus weights and body weight relative spleen weights were markedly lower than Controls in both sexes at 16000/8000 ppm and in females at 8000/4000 ppm. Body weight relative brain weights were higher than Control in both sexes at 16000/8000ppm.

Organ Weights - F1 generation
At scheduled termination of the Cohort 1A animals, the mean terminal body weight of the males and females given 8000/10000/16000 ppm and males given 4000/5000/8000 ppm were statistically significantly lower than Controls, reflecting the inferior body weight performance previously discussed. Differences in absolute organ weights, and body weight relative brain weights reflected the lower terminal body weights recorded, and no direct effect of treatment was inferred.

Analysis of organ weights revealed, when compared to Controls, increased body weight relative kidney weights in all groups of treated males and increased liver weights in males and females given 4000/5000/8000 ppm or 8000/10000/16000 ppm, with a dose response relationship apparent and with differences from Control attaining statistical significance.

At scheduled termination of the Cohort 1B animals, the mean terminal body weight of the males and females given 8000/10000/16000 ppm was statistically significantly lower than Controls, reflecting the inferior body weight performance previously discussed. The weight of the reproductive organs was considered unaffected by treatment. Differences in absolute organ weights, and body weight relative testes weights reflected the lower terminal body weights recorded, and no direct effect of treatment was inferred.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At scheduled termination of the Cohort 1A animals at approximately 13 weeks of age, when compared to Controls there was an increased incidence of dilated renal pelvis in all groups of treated males, although in the absence of a dose response relationship and a similar finding was not observed in Cohort 1B males at approximately 22 weeks of age. There were no other macroscopic abnormalities detected among the Cohort 1A animals which were clearly attributable to D,L-Menthol administration.

At scheduled termination of the Cohort 1B animals at approximately 22 weeks of age for males and on Day 28 of lactation for females, when compared to Controls there were no macroscopic abnormalities detected which were clearly attributable to D,L-Menthol administration.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

Histopathology - Cohort 1A

Treatment Related Findings
Changes related to treatment with D,L-Menthol were seen in the kidneys of males and the liver of females in the F1 Cohort 1A animals.
 
Kidneys
In males, an increased incidence and severity of hyaline droplet accumulation was seen with associated changes; multifocal tubular basophilia was present in animals given D,L-Menthol at all dietary levels, with cast formation apparent at the highest dietary level.

Minor increases in pelvic dilation were seen in all groups of treated males but this did not occur in a dose dependent manner and was not present in females and thus is considered unlikely to be related to treatment.

Summary of treatment related findings in the kidneys for Cohort 1A males killed after completion of treatment
Group/sex 1M 2M 3M 4M
Dietary level (ppm) 0 2000/ 4000/ 8000/
2500/ 5000/ 10,000/
4000 8000 16000

Accumulation, Hyaline Droplets
Minimal 5 10 4 4
Slight 1 10 14 10
Moderate 0 0 2 6
Total 6 20 20 20

Basophilia, Tubular, Multifocal
Minimal 0 4 11 4
Slight 0 1 3 7
Moderate 0 0 0 1
Total 0 5 14 12

Cast(s)
Minimal 1 0 0 2
Total 1 0 0 2

Dilatation, Pelvic
Minimal 2 1 3 3
Slight 0 2 3 1
Moderate 0 3 0 1
Total 2 6 6 5

Number of tissues examined 20 20 20 20

Liver
Centrilobular hypertrophy of minimal to slight degree was present in the liver of all females given D,L-Menthol at 8000/10000/16000 ppm. It was also present at minimal degree in a few animals given 4000/5000/8000 ppm and two given 2000/2500/4000 ppm.
 
Summary of treatment related findings in the liver for Cohort 1A females killed after completion of treatment
Group/sex 1F 2F 3F 4F
Dietary level (ppm) 0 2000/ 4000/ 8000/
2500/ 5000/ 10000/
4000 8000 16000

Hypertrophy, Centrilobular
Minimal 0 2 10 12
Slight 0 0 0 8
Total 0 2 10 20
Number of tissues examined 20 20 20 20

Incidental Findings
All other histological changes were considered to be unrelated to treatment.

Seminiferous tubules of the testes were evaluated with respect to their stage in the spermatogenic cycle and the integrity of various cell types present within the different stages. No cell or stage specific abnormalities were noted. The qualitative examination of the ovary and staging of the estrus cycle revealed no abnormality.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Ovarian Follicle Counts and Corpora Lutea - Cohort 1A
F1 Cohort 1A females given 8000/10000/16000 ppm showed a statistically significant increase in follicle counts and a slight increase in corpora lutea counts when compared to Controls.

Sperm Assessment - Cohort 1A
At scheduled termination of the Cohort 1A males, the motility and motion of the sperm, testicular and cauda epididymal sperm counts and sperm morphology were considered unaffected by treatment with D,L-Menthol at all dietary concentrations investigated.
 
Vaginal Opening - Cohort 1A
Cohort 1A females were assessed daily from the day of vaginal opening until the first estrus smear was observed. There was no clear effect of treatment on the interval between vaginal opening and first estrus, although it was noted that one female given 4000/5000/8000 ppm and two females given 8000/10000/16000 ppm achieved first estrus slightly later than the latest Control female.

Estrous Cycles - Cohort 1A
Estrous cycle regularity was assessed for the Cohort 1A females for 15 days from approximately 75 days of age; no effect of treatment with D,L-Menthol was apparent.

Estrous cycles at termination of the Cohort 1A females were unaffected by treatment at all dietary concentrations.

Pre-Coital Interval, Mating Performance and Fertility, Gestation Length and Gestation Index - Cohort 1B
There was no effect of D,L-Menthol administration on pre-coital interval of the Cohort 1B animals, with all mating pairs showing positive mating evidence at the first estrus opportunity. Mating performance and fertility, and gestation length and gestation index were unaffected at all dietary concentrations investigated.

Stage of Estrous Cycle at Termination - Cohort 1B
Estrous cycles at termination of the Cohort 1B females were unaffected by treatment at all dietary concentrations.

Incidental Findings
There were no incidental findings detected. All other histological changes were considered to be unrelated to treatment.

Seminiferous tubules of the testes were evaluated with respect to their stage in the spermatogenic cycle and the integrity of various cell types present within the different stages. No cell or stage specific abnormalities were noted. The qualitative examination of the ovary and staging of the estrus cycle revealed no abnormality.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

no effects observed

Description (incidence and severity):

The administration of D,L-Menthol at dietary concentrations up to and including 16000 ppm was considered to have no observable effects on the immunophenotyping parameters measured in spleen leukocytes of the Cohort 1A F1 animals.

Effect levels (F1)

1

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

>= 512 - <= 611 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Generation:

F1a

Effect level:

> 4 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

3

Key result

Dose descriptor:

LOAEL

Generation:

F1a

Effect level:

8 000 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

sexual maturation

4

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 4 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Target system / organ toxicity (F1)

1

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

512 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

2

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

2 000 ppm

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Results: F2 generation

General toxicity (F2)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs observed among the F2 offspring that were considered to be related to parental treatment with D,L-Menthol.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The birth weight of male and female F2 offspring was essentially similar in all groups.

In the 8000/10000/16000/8000 ppm group, group mean body weight gain of male and female offspring from Day 4 of age was statistically significantly lower than Control, such that group mean absolute body weight on Day 21 of age was 17-18% lower than Control.

At 4000/5000/8000/4000 ppm, offspring body weight gain was unaffected during Days 1-14 of age, however during Days 14-21 of age mean body weight gain was lower than Control, with statistical significance attained for the differences. Consequently, mean absolute body weights on Day 21 of age were 7-8% lower than Control in both sexes.

Offspring body weight gain at 4000/2000 ppm was unaffected by treatment.

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

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

There was no evidence of an effect of parental D,L-Menthol administration on the ano-genital distance of the F2 offspring on Day 1 of age.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

All F2 male offspring were examined on Day 13 of age and on Day 21 of age for the presence of nipples (areolae). There were no cases of nipples (areolae) being apparent in male offspring in the F2 Control litters on either assessment occasion.

In the 2000/2500/4000/2000 ppm group two male offspring in one litter (Litter 714) were found to have nipples on Day 13 of age. In the 4000/5000/8000/4000 ppm group, 18 male offspring (in a total of 7 litters) were found to have nipples and 48 male offspring in the 8000/10000/16000/8000 ppm group (in a total of 15 litters were found to have nipples).

None of the afore-mentioned male offspring were found to have nipples present on Day 21 of age, demonstrating that the retention of nipples on Day 13 of age was a transient finding.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At scheduled termination on Day 22 of age, the group mean terminal body weight of male and female offspring in the 8000/10000/16000/8000 ppm group and of female offspring in the 4000/5000/8000/4000 ppm group were lower than Control, reflecting the lower body weight gain observed prior to weaning.

Group mean absolute and body weight relative spleen weights were statistically significantly low in male offspring in the 4000/5000/8000/4000 ppm group and in both sexes in the 8000/10000/16000/8000 ppm group. Absolute liver weights were also statistically significantly low among females in the 8000/10000/16000/8000 ppm group.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related macroscopic abnormalities detected among F2 offspring killed or dying prematurely, among F2 offspring culled on Day 4 of age or among the unselected F2 offspring at scheduled termination on Day 22 of age.

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

F2 Litter Responses
One Control female (No. 684) and one intermediate dose female (No. 732) failed to litter, and one low dose female (No. 701), two intermediate dose females (No’s. 725 and 740) and two high dose females (No’s. 741 and 751) were not pregnant. Therefore, the following assessment is made on 19, 19, 17 and 18 litters at 0, 2000/2500/4000/2000 ppm, 4000/5000/8000/4000 ppm and 8000/1000/16000/8000 ppm, respectively.

Litter Size, Survival Indices and Sex Ratio
There was no effect of D,L-Menthol administration on the mean number of uterine implantation sites in the Cohort 1B females at any dietary inclusion level investigated. However, at 8000/10000/16000/8000 ppm the post implantation survival and live birth indices were slightly low such that live litter size on Day 1 of age was 1.8 pups/litter lower than Control.

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

Key result

Dose descriptor:

NOEL

Generation:

F2

Effect level:

>= 512 - <= 611 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Target system / organ toxicity (F2)

Critical effects observed:

not specified

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Formulation Analysis

The mean concentrations of D,L-Menthol in test diets analyzed during the course of the study were within +10/-15% of the nominal concentration, confirming the accuracy of formulation, with the exception ofLast Week (F1 generation) for Group 3, when the males were receiving 8000 ppm, which was-21.0% from nominal concentration. Contingency analysis was not performed as samples were outside of the achieved stability period. Considering that all other analytical occasion results were within specification, including all groups of females in the final week, and that homogeneity and stability of the test item in the diet had previously been confirmed for 24 days, it is considered that the formulation method was fit for purpose, the diets were prepared correctly throughout the study and despite this isolated incidence it is considered that the animals received the intended exposure.

The difference from mean remained within 5%, confirming precise analysis. The procedural recoveries remained within the validated range, confirming the continued accuracy of the analytical procedure.

F0 Generation

Achieved Dose

The overall mean achieved dose during Weeks 1-18 of treatment period was 247, 499 and 1016 mg/kg/day for males at 4000, 8000 and 16000 ppm, respectively.

The overall mean achieved dose during Weeks 1-10 of the pre-pairing treatment period was 300, 594 and 1205 mg/kg/day for females at 4000, 8000 and 16000 ppm, respectively.

The overall mean achieved dose during Days 0-20 of gestation was 260, 520 and 1029 mg/kg/day for females at 4000, 8000 and 16000 ppm, respectively.

The overall mean achieved dose during Days 1-14 of lactation was 303, 599 and 1184 mg/kg/day for females at 2000, 4000 and 8000 ppm, respectively.

F1 Litter Responses

Two Control females (No’s. 215 and 219) and one low dose female (No. 241) were not pregnant, and one female in the intermediate dose group (No. 264) was killed for welfare reasons on Day 1 of lactation. Therefore, the following assessment is made on 23, 24, 24 and 25 litters at 0, 4000/2000, 8000/4000 and 16000/8000 ppm, respectively.

General Condition of Offspring

There were no clinical signs observed among the F1 offspring that were considered to be related to parental treatment with D,L-Menthol.

Litter Size, Survival Indices and Sex Ratio

In the 16000/8000 ppm group, the mean number of uterine implantation sites was statistically significantly lower than Control. As a consequence, the total offspring born and mean number of live offspring on Days 1 and 4 of age (before litter standardisation) was also lower than Control. There was no effect of D,L-Menthol on mean litter size at 8000/4000 or 4000/2000 ppm.

Offspring survival indices to Day 21 of age and sex ratio were unaffected by D,L-Menthol administration at all dietary concentrations investigated.

Offspring Macropathology

There were no test item-related macroscopic abnormalities detected among F1 offspring killed or dying prematurely, among F1 offspring culled on Day 4 of age or among the unselected F1 offspring at scheduled termination on Day 22 of age.

F1 Generation

 Achieved dose

The overall mean achieved dose from Day 21 of age to Day 131 of the formal F1 generation period for males was 203, 419 and 837 mg/kg/day for males in Groups 2 to 4, respectively.

The overall mean achieved dose from Day 21 of age to Day 71 of the formal F1 generation (end of Cohort 1A/pre-pairing period for Cohort 1B) was 229, 455 and 892 mg/kg/day for females in Groups 2 to 4, respectively.

The overall mean achieved dose during Days 0-20 of gestation was 259, 512 and 1039 mg/kg/day for females at 4000, 8000 and 16000 ppm, respectively.

The overall mean achieved dose during Days 1-14 of lactation was 294, 611 and 1235 mg/kg/day for females at 2000, 4000 and 8000 ppm, respectively.

Applicant's summary and conclusion

Conclusions:

The purpose of this extended one generation reproductive toxicity study was to assess the influence of D,L-Menthol, an industrial chemical, on reproductive performance when administered continuously in the diet to CD rats at dietary inclusion levels up to and including 16000 ppm. The evaluation included assessment of the integrity and performance of the adult male and female reproductive tract, and systemic toxicity in pregnant and lactating females and in young and adult offspring. In addition, an evaluation of the maturing reproductive tract and its integrity and function was performed.

Based on the results obtained in this study it was concluded that the No-Observed-Effect-Level (NOEL) for reproductive performance of the F0 and F1 Cohort 1B animals was the intermediate dose of 419-499 mg/kg/day for males and 455-594 mg/kg/day for females due to lower litter size observed in both generations at the high dose level, a level which was associated with reduced food consumption and body weight gain in the parental animals of both generations.

Histopathological changes in the kidney of F0 and F1 Cohort 1A males, although of no relevance to man, were adverse within the context of this study and therefore a No-Observed-Adverse-Effect-Level (NOAEL) for the kidney was not established. Aside from the kidney, the NOAEL for systemic toxicity in the F0 and F1 adult animals was concluded to be the intermediate dose of 419-499 mg/kg/day for males and 455-594 mg/kg/day for females, based upon the impaired body weight gain at the high dose level.

The NOEL for the F1 and F2 offspring up to weaning was concluded to be the intermediate dose of 512-611 mg/kg/day due to reduced pre-weaning growth in both generations.
 

Executive summary:

The purpose of this study was to assess the influence of D,L-Menthol on reproductive performance when administered continuously in the diet to CD rats. The evaluation included assessment of the integrity and performance of the adult male and female reproductive tract, and systemic toxicity in pregnant and lactating females and in young and adult offspring. In addition, an evaluation of the maturing reproductive tract and its integrity and function was performed.

In the F0 generation, three groups of 25 male rats received D,L-Menthol orally, via the diet, at concentrations of 4000, 8000 or 16000 ppm for ten weeks before pairing and until termination. In addition, three groups of 25 female rats received D,L-Menthol orally, via the diet, at concentrations of 4000, 8000 or 16000 ppm for ten weeks before pairing, throughout pairing and gestation, and at concentrations of 2000, 4000 or 8000 ppm during lactation. Corn oil was added to the premix as a stabilizer at a ratio of 5:1 test item:corn oil. A similarly constituted Control group received untreated basal diet with added corn oil for the same duration. 

In theF1generation, 40 males and 40 females were treated from weaning to their scheduled termination (relevant to each cohort). From weaning to Week 4 of the formal F1 generation the dietary concentrations were 2000, 4000 and 8000 ppm. For Weeks 5 and 6 of the formal F1 generation the dietary concentrations were 2500, 5000 and 10000 ppm. From Week 7 of the formal F1 generation until termination the dietary concentrations were 4000, 8000 and 16000 ppm, with the exception of the lactation period for the Cohort 1B females when the dietary concentrations were 2000, 4000 and 8000 ppm. A similarly constituted Control group received untreated basal diet with added corn oil for the same duration. 

The dietary concentrations employed were selected in an attempt to achieve an equivalent high dose level of 1000 mg/kg bw/day. Diet concentrations were reduced during lactation to compensate for lactation-specific increases in food consumption. Similarly, during the early stages of the formal F1 generation, the dietary concentrations were steadily increased from lactation levels to adult levels in order to avoid excessively high achieved dose levels.

Clinical observations, body weight, food consumption and macropathology examinations were performed on all animals for signs of toxicity, with special emphasis on the integrity and performance of the male and female reproductive system and the health (including thyroid hormone analysis, urinalysis and immunophenotyping in spleen lymphocytes), growth, development and function of the offspring.

At weaning, two cohorts of selected males and females were assigned for further investigations, including sexual maturation and reproductive organ integrity and function.

The cohorts were as follows:

Cohort	Designation	Animals/Cohort	Sexual maturation assessment	Approximate age at necropsy
1A	Reproductive & general toxicity	20M + 20F	Yes	13 weeks
1B	Reproductive toxicity	20M + 20F	Yes	23 weeks

 

 Results

There was no effect of D,L-Menthol administration at any dietary inclusion level investigated on serum T4 or TSH concentrations in F0 or F1 Cohort 1A adult animals, or in the F1 offspring on Day 22 of age.

D,L-Menthol administration at dietary inclusion levels up to and including 16000 ppm was well tolerated by F0 and F1 animals throughout the study, with no test item-related premature deaths or test item‑related changes in general clinical condition, and estrous cycle regularity of the F0 and the F1 Cohort 1A females, and pre-coital interval, mating performance, conception rate and fertility index of the F0 and F1 Cohort 1B animals were unaffected. Similarly, there was no effect of treatment on the duration between vaginal opening and first estrus in the F1 Cohort 1A females or the stage of estrus at termination of the F0 or the F1 Cohort 1A and 1B females, haematological parameters in F0 and F1 Cohort 1A animals or sperm motility/counts/morphology of the F0 and F1 Cohort 1A males. Macroscopic examination of the F0 animals, the F1 Cohort 1A females and F1 Cohort 1B animals at scheduled termination did not reveal any test item-related abnormalities. There were also no observable effects on the immunophenotyping parameters measured in spleen leukocytes of the F1 Cohort 1A animals.

Kidney weights were increased at the end of the treatment period at all dietary inclusion levels in F0 and F1 Cohort 1A males, which occurred concurrently with an increased but non-dose-dependent incidence of dilated renal pelvis observed macroscopically in F1 Cohort 1A males. The cause of this increased kidney weight was identified at the histopathological examination, by the presence of minimal to moderate hyaline droplet accumulation. The increase in hyaline droplets in the kidneys of males is consistent with the accumulation of α2µ‑globulin, a common finding in untreated male rats, as indeed seen in Control F1 Cohort 1A males at a low incidence. Hyaline droplet accumulation is specific to the male rat and is generally not considered to be significant in man. However, clearance or accumulation of α2µ‑globulin can be altered when it is bound to a xenobiotic, and accumulation of this complex can lead to tubular damage and this is described as α2µ‑globulin nephropathy, in this study manifest as minimal/moderate multifocal tubular basophilia and the formation of casts. Diffuse or multifocal tubular dilation was also present in males given 8000 or
16000 ppm in the F0 generation only and this indicates tubular changes associated with D,L‑Menthol; this finding is not directly associated α2µ‑globulin nephropathy and therefore may be of toxicological significance. The α2µ‑globulin is a protein synthesized by the liver of male rats only and therefore its accumulation and subsequent tubular damage is both sex and species specific. In the context of this study, the hyaline droplet accumulation and subsequent associated pathology were considered adverse in the animals affected, since degenerate changes were also present, but, due to the reasons specified previously, such findings have long been considered irrelevant in humans. These findings occurred at all administered dietary inclusion levels, and generally showed a dose-dependent increase in incidence/severity. There were some minor changes in the blood plasma of the males, with increase in urea concentrations in F0 and F1 Cohort 1A males and increase in calcium concentration in F1 Cohort 1A males, along with a slight increase in acidity of the urine of F1 Cohort 1A males that may be consequences of hyaline droplet accumulation. There was a decrease in plasma creatinine in F0 and F1 Cohort 1A females and an increase in plasma sodium and calcium concentrations in F0 females which would possibly indicate minor alterations in renal function due to excretion of the test item and/or its metabolites, with increased kidney weights also apparent in F0 females, although there was no associated histopathological finding in these females and the subtle findings in females were therefore considered not to be adverse.

Centrilobular hypertrophy (minimal/slight) in the liver occurred at an increased incidence in F0 and F1 Cohort 1A females at all dietary inclusion levels, and correlated with a slight organ weight increase. These hypertrophic changes are suggestive of an adaptive response to mixed function oxidase induction in the liver, but the precise mechanism of hypertrophy cannot be determined by histopathological examination alone (Sahota et al, 2013). The change identified in this study was of mild or minimal severity, limited to females and was not accompanied by other test item-related changes in the liver. There was no histopathological correlate for the slight, but statistically significant and dose-dependent increase in liver weight consistently observed in both generations of F0 and F1 Cohort 1A males.

There was no effect of parental D,L-Menthol administration on F1 or F2 offspring birth weight, ano-genital distance, sex ratio, survival after Day 1 of age to weaning or clinical condition. When compared to Controls, for F1 litters in the 16000/8000 ppm group, the mean number of uterine implantation sites was statistically significantly low, resulting in a lower total number of offspring born and subsequently live offspring on Day 1 and Day 4 of age (before litter standardization). Whilst differences in these litter parameters in the 8000/10000/16000/8000 ppm group F2 litters did not attain statistical significance, mean values of litter size in the high dose F1 and F2 litters were lower than all other study groups in both generations, and five Control groups from three OECD 443 studies conducted during 2017-2019 (range of mean live litter size, 13.2 to 15.8 pups; mean 14.6 pups). Consequently, the lower number of uterine implantation sites and subsequently low litter size in the high dose group in both generations were considered test item-related and adverse.

In both generations, offspring growth was reduced from Day 4 of age to weaning in the high dose litters, with absolute body weight at weaning of the F1 and F2 litters on Day 21 of age being 17-18% lower than Control; the extent of the reduction in offspring growth in the high dose group in both generations was considered to be adverse. Offspring growth was also slightly low in the intermediate dose litters in both generations, from Day 7 of age in the F1 litters and from Day 14 of age in the F2 litters such that absolute body weight was 6-8% lower than Control at weaning. There findings were seen together with decreased food consumption in the dams and/or offspring and although test item-related were considered not to be adverse at the extent observed.

All F1 and F2 male offspring were assessed on Day 13 of age for the presence of nipples (areolae). In both generations, a dose dependent high incidence of male offspring in the intermediate and high dose groups showed at least one pair of areolae, with the incidence being notably higher in the F2 male offspring than in the F1 male offspring. The nipples were no longer apparent when F1 males were re-assessed in Week 5 of the formal F1 generation or when F2 males were re-assessed on Day 21 of age, the retention of nipples on Day 13 of age, although clearly related to parental D,L-Menthol administration, was a transient finding with no subsequent effects on the weight/histopathology of the male reproductive organs, fertility, mating performance or sperm analysis, and therefore not adverse.

Absolute and body weight relative spleen weights were low in F1 and F2 offspring in the intermediate and high dose groups on Day 22 of age. These differences were considered to be of no toxicological significance since there were no histopathological changes in the spleen of F0 or F1 Cohort 1A animals, or effects on spleen immunophenotyping in the F1 Cohort 1A animals. The other minor differences in unselected F1 and F2 offspring organs weights were attributable to the lower terminal body weights recorded. There were no test item-related macroscopic abnormalities detected among F1 or F2 offspring dying prematurely or among F1 or F2 offspring killed at scheduled termination.

A 3.2-day delay in attainment of balano pre-putial separation (BPS) occurred in selected F1 males in the 8000/10000/16000 ppm group, which occurred in the absence of an effect on absolute body weight on the day of attainment. Whilst a relationship to treatment for this delay cannot be excluded, BPS was achieved when comparable body weights were attained in all F1 males indicating that this observation may be secondary to reductions in body weight gain, and in the absence of subsequent effects on the weight/histopathology of the male reproductive organs, fertility, mating performance or sperm analysis, the delay in attainment of sexual maturation was considered not to be adverse.

The slight increase in ovarian follicle and corpora lutea counts in F1 Cohort 1A females given 8000/10000/16000 ppm was considered to be of no toxicological significance in view of the absence of any histopathological change in the ovaries or associated increases in litter size in the F1 Cohort 1B females.

Biochemical analysis of the plasma at scheduled termination of the F0 and F1 Cohort 1A animals revealed when compared to Controls, a slight increase in gamma glutamyl transpeptidase concentrations in high dose animals in both generations and in intermediate group F1 Cohort 1A females, low glucose concentrations in low dose F0 males and in intermediate and high dose males and females in the F0 generation, and slightly high cholesterol concentrations in high dose F1 Cohort 1A animals, and slightly low albumin/globulin ratio (due to slight changes in total protein and albumin concentrations) in high dose animals of both generations. In F0 and F1 Cohort 1A females, these biochemical changes were considered to be related to the aforementioned hypertrophy observed in the liver at histopathological examination, and were considered to be non-adverse at the degree observed. In the F0 and F1 Cohort 1A males, there were no histopathological correlates for these minor biochemical changes, therefore these differences were considered to be of no toxicological significance.

Conclusion

The purpose of this extended one generation reproductive toxicity study was to assess the influence of D,L-Menthol, an industrial chemical, on reproductive performance when administered continuously in the diet to CD rats at dietary inclusion levels up to and including 16000 ppm. The evaluation included assessment of the integrity and performance of the adult male and female reproductive tract, and systemic toxicity in pregnant and lactating females and in young and adult offspring. In addition, an evaluation of the maturing reproductive tract and its integrity and function was performed.

Based on the results obtained in this study it was concluded that the No-Observed-Effect-Level (NOEL) for reproductive performance of the F0 and F1 Cohort 1B animals was the intermediate dose of 419-499 mg/kg/day for males and 455-594 mg/kg/day for females due to lower litter size observed in both generations at the high dose level, a level which was associated with reduced food consumption and body weight gain in the parental animals of both generations.

 

Histopathological changes in the kidney of F0 and F1 Cohort 1A males, although of no relevance to man, were adverse within the context of this study and therefore a No-Observed-Adverse-Effect-Level (NOAEL) for the kidney was not established. Aside from the kidney, the NOAEL for systemic toxicity in the F0 and F1 adult animals was concluded to be the intermediate dose of 419-499 mg/kg/day for males and 455-594 mg/kg/day for females, based upon the impaired body weight gain at the high dose level.

 

The NOEL for the F1 and F2 offspring up to weaning was concluded to be the intermediate dose of 512-611 mg/kg/day due to reduced pre-weaning growth in both generations.