4-isopropyl-m-cresol

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 August 2014 - 25 August 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well conducted and well described study in accordance with GLP and OECD Guideline 422 without any deviation.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP compliance programme (inspected on July 01-03, 2014 / signed on September 16, 2014)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-isopropyl-m-cresol

EC Number:

221-761-7

EC Name:

4-isopropyl-m-cresol

Cas Number:

3228-02-2

Molecular formula:

C10H14O

IUPAC Name:

4-isopropyl-m-cresol

Test material form:

other: solid

Details on test material:

- Name of test material (as cited in study report): Parathymol P16029 (Xylenol)
- Physical state: White solid
- Analytical purity: 99.77% (Pre-study chemistry); 99.68% (Live animal phase)
- Purity test date: 24 July 2013
- Lot/batch No.: 1732349 (Pre-study chemistry); 100039076 (Live animal phase)
- Expiration date of the lot/batch: 1 July 2015 (Pre-study chemistry); 31 March 2016 (Live animal phase)
- Storage condition of test material: At ambient temperature

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited
- Age of animals at study initiation: Approximately 10 weeks
- Weight at study initiation:: Males: 328-391 g; Females: 236-296 g
- Housing (Number of animals per cage): Pre pairing - up to five animals of one sex; During pairing - one male and one female; Males after mating - up to five animals; Gestation - one female; Lactation: one female + litter. Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatisation, gestation, littering, lactation and maturation periods. Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
- Diet: SDS VRF1 Certified powdered diet, ad libitum
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum
- Acclimation period: 6 days

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

IN-LIFE DATES: 28 August 2014 - 25 August 2015

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: SDS VRF1 certified diet

Details on exposure:

DIET PREPARATION
- Diet: SDS VRF1 certified diet
- Method of preparation: The required amount of test material was ground to a powder using a mortar and pestle, and was then added to an approximately equal amount of sieved diet and stirred. An amount of sieved diet approximately equal to the weight of the mixture was added and the mixture stirred until it appeared homogeneous. This doubling up process with the sieved diet was continued until approximately half of the premix diet had been added. At this stage, the mixture was ground using a mechanical grinder. Coarse diet was then added to make the premix up to the required amount and mixing was continued in a Turbula mixer for 200 revolutions. This ensured the test substance was dispersed in the diet. The premix was then diluted with further quantities of coarse basal diet to prepare the required concentration for each test group.
- Correction factor: None, used as supplied.
- Frequency of preparation: Weekly
- Storage of formulation: The stability of a homogenous formulation of the substance in the vehicle was demonstrated over a period of up to:
1000 ppm: 24 hours at room temperature; 1000 ppm: 22 days at -20 °C; 20000 ppm: 22 days at room temperature or -20 °C. Therefore dose was prepared weekly and frozen until required for feeding and changed daily within the food hopper.

Details on mating procedure:

- M/F ratio per cage: 1: 1
- Pairing commenced: After 2 weeks of treatment for males and females
- Length of cohabitation: Up to 2 weeks
- Proof of pregnancy: Presence of sperm within the vaginal smear and/or ejected copulation plugs referred to as Day 0 of gestation.
- Male/female separation: Day when mating evidence was detected.
- Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity: Before commencement of treatment, the suitability of the proposed mixing procedure was determined and specimen formulations were analysed to assess the stability and homogeneity of the test substance in the diet matrix.
Achieved concentration: Samples of each formulation prepared for administration in Weeks 1 and 5 of treatment were analysed for achieved concentration of the test substance.

Duration of treatment / exposure:

Males: Two weeks pre-pairing up to necropsy after minimum of five weeks.
Females: Two weeks before pairing, then throughout pairing and gestation until Day 6 of lactation.
Animals of the F1 generation were not dosed.

Frequency of treatment:

Continuously. Diet in food hoppers was changed daily

Doses / concentrations

Remarks:

Doses / Concentrations:
1500, 4500 and 15000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dose levels were selected in conjunction with the Sponsor following the review of data from a 14 day preliminary study at this laboratory (Envigo Study Number BEZ0001). In that study Parathymol P16029 was well tolerated at dose levels of 1500, 4500 and 15000 ppm. Up to day 4 of treatment effects included body weight loss and low food consumption at 15000 ppm and in males only at 4500 ppm low body weight gain (Days 1-4). After day 4 food consumption was high for males and females and body weight gain was reduced for males only at 15000 ppm. At both 15000 and 4500 ppm water consumption was high and at necropsy there was a suggestion of increased body weight relative kidney weights in males. None of the effects observed were considered adverse and none precluded the use of dose levels up to and including 15000 ppm in a subsequent OECD 422 study.
In this study therefore a high dose of 15000 ppm, expected to result in an achieved dose level of 1000 mg/kg bw/day was selected as the limit dose level for the OECD 422 study type and dose levels of 1500 and 4500 ppm were selected to demonstrate a dose response of any effects observed.
- Rationale for animal assignment: On arrival and non-selective allocation to cages.
On Day 1 of study all animals were weighed and body weights were reviewed before dosing commenced to ensure any variation in body weight of animals did not exceed ±20% of the mean for each sex.

Positive control:

Not applicable

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of ill-health amongst the occupant(s). During the acclimatisation period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced and during each week of treatment and on Days 0, 6, 13 and 20 after mating and Days 1 and 6 of lactation, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period except during pairing or for females after mating or during lactation), by an observer unaware of the experimental group identities.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule:
Sensory reactivity and grip strength assessments were performed on the five lowest numbered surviving males in each group during Week 5 of treatment and on the five lowest numbered surviving lactating females (excluding female 54) in each group on Days 4-6 of lactation.
Motor activity: During Week 5 of treatment for males and on Days 4-6 of lactation for females, the motor activity of the five lowest numbered surviving males and the five lowest numbered lactating females (excluding female 54) in each group.

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 males: Before dosing on the day that treatment commenced (Week 0) and weekly thereafter
F0 females: Before dosing on the day that treatment commenced (Week 0), weekly before pairing; Days 0, 6, 13 and 20 after mating; Day 1, 4, and 7 of lactation.

FOOD CONSUMPTION: Yes
- Time schedule for examinations:
F0 animals: Daily, from the day that treatment commenced (Day 1).
Food consumption was not recorded for males and females during the period when paired for mating, from Days 15, and recommenced for males on Day 22. For females after mating food consumption was recorded daily.
From these records the mean daily consumption per animal (g/animal/day) was calculated for each phase.

WATER CONSUMPTION: Yes
- Fluid intake was assessed by daily visual observation.

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 2 prior to pairing. The five lowest numbered surviving males and females per group
- Animals fasted: Yes, overnight fasting
- Animals were held under light general anaesthesia induced by isoflurane. Blood samples were withdrawn from the sublingual vein.
- Haematology parameters: Haematocrit, Haemoglobin concentration, Erythrocyte count (RBC), Absolute reticulocyte count, Percentage reticulocyte count, Mean cell haemoglobin, Mean cell haemoglobin concentration, Mean cell volume, Red cell distribution width, Total leucocyte count, Differential leucocyte count: Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells, Platelet count, Morphology: Anisocytosis, Macrocytosis, Microcytosis, Hypochromasia, Hyperchromasia, Prothrombin time and Activated partial thromboplastin time.
- Blood Chemistry parameters: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Total bilirubin, Bile acids, Urea, Creatinine, Glucose, Total cholesterol, Triglycerides, Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus, Total protein, Albumin and Albumin/globulin ratio (A/G Ratio).

PARTURITION OBSERVATIONS AND GESTATION LENGTH:
- Duration of gestation: Time elapsing between the detection of mating and commencement of parturition.
- Parturition observations: From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.

Oestrous cyclicity (parental animals):

Wet smears: After pairing until mating, using pipette lavage

Sperm parameters (parental animals):

No data

Litter observations:

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

Postmortem examinations (parental animals):

SACRIFICE
- F0 males: After Week 5 investigations completed.
- F0 females: Day 7 of lactation
- Method of sacrifice: F0 animals were killed by Carbon dioxide asphyxiation with subsequent exsanguination.

GROSS NECROPSY
- All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative. In each uterine horn, number of implantation sites was recorded.
ORGAN WEIGHTS
- For bilateral organs, left and right organs were weighed together, unless specified above. Requisite organs were weighed for animals killed at scheduled intervals.

HISTOPATHOLOGY / ORGAN WEIGHTS
- The organs weighed, tissue samples fixed and sections examined microscopically are detailed in table 7.8.1/1 and 7.8.1/2.
- Fixation: Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:
Testes: Initially in modified Davidson’s fluid; Eyes: In Davidson’s fluid.
For all animals examined, samples of any abnormal tissues were retained. In those cases where a lesion was not clearly delineated, contiguous tissue was fixed with the grossly affected region.
- Histology
Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
Full List: All animals killed or dying prematurely.
The five lowest numbered surviving males and females (with a live litter) in Groups 1 and 4 at scheduled termination.
Abnormalities: All animals
Thymus: The five lowest numbered surviving females (excluding female 54) (with a live litter) in Groups 2 and 3 at scheduled termination.
Routine staining: Sections were stained with haematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid/Schiff (PAS) method.
- Light microscopy: Tissues preserved for examination were examined as follows:
Groups 1 and 4: Five lowest numbered surviving males and females (with a live litter), at scheduled termination - All specified in table 7.8.1/1 and 7.8.1/2
All F0 adult animals: Abnormalities
Groups 2 and 3: Five lowest numbered surviving females (excluding female 54) (with a live litter), at scheduled termination - Thymus

Postmortem examinations (offspring):

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

GROSS NECROPSY
- Offspring before scheduled termination: Subject to a complete internal and external macroscopic examination (including an assessment of stomach milk content) where possible.
- Offspring at scheduled termination: Subject to an external macroscopic examination. Only externally abnormal offspring were examined internally.

Statistics:

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

Reproductive indices:

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

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

Offspring viability indices:

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

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- Clinical signs were considered minor in nature and did not indicate any direct reaction to Parathymol P16029.
- There were no premature deaths.

BODY WEIGHT (PARENTAL ANIMALS)
- Mean body weight change for males receiving 15000 ppm was lower than Controls during the first four weeks of treatment, attaining statistical significance for Weeks 0-2 and 3-4. Subsequent overall mean body weight gain for the entire treatment period (Weeks 0-5) was significantly lower than Controls (68% of Controls). Male body weight performance at 4500 or 1500 ppm was similar to Controls and unaffected by treatment.
- Body weight loss was recorded for females receiving 15000 ppm during week 1 of study and during Weeks 1-2 body weight gain was lower than Controls such that overall body weight loss was observed for the period before pairing (Weeks 0-2), compared to overall mean body weight gain in all other groups. During gestation at this dose level, lower body weight gain than Controls was observed, attaining statistical significance throughout. Mean body weight loss was observed during Days 1-4 of lactation, compared to weight gain in all other groups, followed by similar weight gain to Controls during Days 4-7 of lactation.
- For females receiving 4500 ppm lower body weight gain than Controls was observed during the period prior to pairing, and slightly low weight gain compared to Controls was observed during Days 0-6 of gestation. Subsequent body weight gain during gestation and lactation was similar, or superior, to Controls.
- At 1500 ppm female body weight gain was similar to Controls during the first week of treatment, but slightly lower than Controls was apparent during the second week before pairing, with mean overall body weight gain prior to pairing being slightly lower than Controls (78% of Controls). Body weight gain during gestation and lactation was similar, or superior, to Controls.

FOOD CONSUMPTION (PARENTAL ANIMALS)
- Food consumption during Week 1 of study was marginally low in males, and slightly low in females receiving Parathymol P16029 at 15000 ppm, this effect was most pronounced during the first 3 or 4 days of treatment. Thereafter food consumption at 15000 ppm for males throughout the remainder of the treatment period, and for females the remainder of the pre pairing period was generally similar to Controls. Females receiving 15000 ppm subsequently showed lower food consumption than Controls during Days 0-5 and 6-12 of gestation (attaining statistical significance) and statistically significant low food consumption throughout lactation.
- At 4500 ppm, food consumption for males was slightly lower than Controls during the first 3 days of treatment, and for females for the first day of treatment only. Thereafter mean values were generally similar to Controls for males throughout the remainder of treatment and for females during the remaining pre-pairing period and gestation. During Days 1-4 of lactation food consumption was low, attaining statistical significance.
- At 1500 ppm food consumption for females was lower than Controls on the first day of treatment only and for the remainder of the pre-pairing period food consumption was similar to Controls. During gestation and lactation, food consumption for females at this dose level was similar to Controls. Male food consumption at 1500 ppm was similar to Controls throughout the entire treatment period.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
- Overall achieved dose levels of Parathymol P16029 for males were 98, 289 and 1038 mg/kg bw/day at 1500, 4500 and 15000 ppm respectively. Achieved dose levels for females were 110, 320 and 1010 mg/kg bw/day before pairing, 104, 323 and 1048 mg/kg bw/day during gestation and 206, 608 and 1888 mg/kg bw/day during lactation.
- Achieved dose generally maintained the intervals between dietary concentrations. During lactation achieved doses were higher reflecting the increased physiological demand on the dams.

WATER CONSUMPTION (PARENTAL ANIMALS)
- Visual water consumption was notably high in animals receiving 15000 or 4500 ppm, with a difference to the Control group noted on 28 occasions and 14 occasions respectively. The three occasions that an effect was noted in the 1500 ppm group is considered not biologically significant. The effects at 15000 ppm influenced the water bottle refilling frequency, which at times, needed to be increased to accommodate this effect.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- There was no effect of Parathymol P16029 on pre-coital interval, fertility or gestation length or gestation index.

ORGAN WEIGHTS (PARENTAL ANIMALS)
- Organ weights adjusted for terminal body weight indicated high kidney and liver weights for males receiving 15000 ppm, and slightly low heart and ovary weights for females at 15000 ppm, attaining statistical significance. Low thymus weight in females receiving 15000 or 4500 ppm was apparent however this did not attain statistical significance.
- There were no differences from Control in the adjusted weights of other organs at 15000 ppm and no effect of treatment upon organ weights at 4500 or 1500 ppm.

GROSS PATHOLOGY (PARENTAL ANIMALS)
- The macroscopic examination performed on the males after 6 weeks of treatment and on the females on Day 7 of lactation revealed no test substance related lesions.
- The incidence and distribution of all findings were consistent with the common background seen at this laboratory.

HISTOPATHOLOGY (PARENTAL ANIMALS)
- Treatment related findings were identified in the thymus.
- Thymus: An increased incidence and severity of thymus involution/atrophy was seen in all females receiving 15000 ppm compared with controls. This finding correlated with the decreased thymus weights recorded at necropsy.

OTHER FINDINGS (PARENTAL ANIMALS)
NEUROBEHAVIORAL EXAMINATION
- Sensory reactivity and grip strength: There was considered to be no reaction to Parathymol P16029 within the sensory reactivity or grip strength assessments performed.
- Motor activity: Motor activity results did not demonstrate any clear differences from the Control group. Assessment of low beam (ambulatory movement) and high beam (rearing activity) for group mean total activity values were similar in all groups assessed.
Occasion periods attained statistical significance however these were isolated, did not demonstrate dose related trends and were apparent in only one sex at the point affected and are therefore considered not to represent a direct effect of treatment.

HAEMATOLOGY
- The haematological examinations during Week 2 of treatment revealed slightly low reticulocyte counts in males and females receiving 15000 ppm, attaining statistical significance in females only. Prothrombin time was extended for males and females at 15000 ppm, attaining statistical significance, and activated partial thromboplastin time was slightly but statistically significantly extended for females only at 15000 ppm. Statistically higher Eosinophil counts were observed in males at 15000 ppm but this was not observed in females at the same dose level.
- At 4500 or 1500 ppm there were no clear differences from Control in haematological parameters that would suggest a relationship to treatment at these dietary inclusion levels.

CLINICAL CHEMISTRY
- A number of differences from controls occurred at 15000 ppm only, including three that were statistically significant, but these were minor, and were inconsistent between the two sexes and therefore attributed to normal variation. Such differences included low sodium in males at 15000 ppm, and low bilirubin and phosphorus levels in females receiving 15000 ppm.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

15 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOAEL

Remarks:

reproductive performance

Effect level:

15 000 ppm

Sex:

male/female

Basis for effect level:

other: The low implantation counts and litter size at 15000 ppm were considered to be associated with the reduced food consumption and not to reflect an intrinsic, specific property of Parathymol” (UN GHS for labelling of chemicals).

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

CLINICAL SIGNS (OFFSPRING)
- Clinical signs of offspring did not indicate any reaction to parental exposure with Parathymol P16029.

LITTER SIZE, SEX RATIO AND SURVIVAL INDICES
- The mean number of implantations and subsequent mean litter size was lower than Controls for females receiving 15000 ppm. Mean numbers of implantations and litter size at 4500 or 1500 ppm were similar to Controls and unaffected by treatment.
- Despite a lower mean number of implantations and live young at 15000 ppm, there was no subsequent effect of treatment upon offspring survival to Day 7 of age at 15000 ppm. Offspring survival was unaffected by treatment at 4500 or 1500 ppm.
- There was no effect of treatment upon mean sex ratio (percentage male offspring) at dietary inclusion levels up to and including 15000 ppm.

BODY WEIGHT (OFFSPRING)
- At 15000 ppm mean body weight of male and female offspring on Day 1 of age were lower than Control, and subsequent body weight gain to Day 7 of age, was also lower than Controls.
- Offspring body weight on Day 1 of age, and subsequent body weight gain to Day 7 of age, at 4500 or 1500 ppm was similar to Controls, and unaffected by treatment.

GROSS PATHOLOGY (OFFSPRING)
- Changes detected at necropsy on offspring killed or dying at or before scheduled termination were part of the normal spectrum expected in this strain of animal, at these ages and at this laboratory.

Effect levels (F1)

Dose descriptor:

NOEL

Generation:

F1

Effect level:

15 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The authors concluded that the low body weight gain in offspring was associated with poor maternal body weight performance, low food consumption and the resultant maternal stress.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Formulation analysis:

The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limit of detection, limit of quantification, linearity of detector response, repeatability, accuracy and precision. Standard and extract stability was not achieved.

The homogeneity was confirmed for Parathymol P16029 in SDS VRF1 certified diet formulations at nominal concentrations of 1000 ppm and 20000 ppm. Stability was confirmed at frozen storage for up to 22 days for both levels, ambient temperature storage for up to 1 day at 1000 ppm and up to 22 days for 20000 ppm.

The mean concentrations of Parathymol P16029, in test formulations analysed for the study were within +10%/-15% of nominal concentrations, confirming accurate formulation. The percentage difference from mean values was within ±3% confirming continued precision of the method.

Applicant's summary and conclusion

Conclusions:

Under the test conditions, the NOAEL of Parathymol P16029 to male and females adult rats for at least 5 weeks was 15000 ppm (equivalent to 1000 mg/kg bw/d). Low implantation site counts impacting subsequent litter size was observed at 15000 ppm. However, the significantly reduced food consumption and reduced bodyweight gain in the females receiving 15000 ppm is considered to be an important factor for the reduced implantations/litter size as described in the literature (Terry et al., 2005) and this interpretation is strengthened by the observation that females receiving 15000 ppm showed involution/atrophy of the thymus which was considered to be indicative of a stress response secondary to the reduced food intake. This was an isolated finding as there was no effect of treatment on mating performance or fertility, the survival of the offspring and no treatment related macroscopic or microscopic changes were detected in the male and female reproductive systems. Therefore, the low implantation counts and litter size at 15000 ppm were considered to be associated with the reduced food consumption and not to reflect an intrinsic, specific property of Parathymol” (UN GHS for labelling of chemicals).

Executive summary:

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted according to OECD Guideline 422 and in compliance with GLP, Parathymol P16029 was administered to groups of Crl:CD(SD) rats (10/sex/dose) at dietary concentrations of 1500, 4500 or 15000 ppm. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 6 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 7 of lactation. The F1 generation received no direct administration of the test substance; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, untreated diet of the same batch. During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, haematology (peripheral blood), blood chemistry, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology investigations were undertaken. Histopathology investigations were undertaken for the first five males and first five females from the Control and high dose groups. The clinical condition, litter size and survival, sex ratio, body weight and macropathology for all offspring were also assessed.

Overall achieved dose levels of Parathymol P16029 for males were 98, 289 and 1038 mg/kg bw/day at 1500, 4500 and 15000 ppm respectively. Achieved dose levels for females were 110, 320 and 1010 mg/kg bw/day before pairing, 104, 323 and 1048 mg/kg bw/day during gestation and 206, 608 and 1888 mg/kg bw/day during lactation.

 

There were no premature deaths; there were no effects on clinical signs, sensory reactivity, grip strength or motor activity.

 

Males receiving 15000 ppm had low mean body weight change during the first four weeks of treatment. Food consumption for these animals was low only for the first three days of study. Male body weight and food performance at 4500 or 1500 ppm was essentially similar to Controls and not adversely affected by treatment.

Females receiving 15000 ppm during week 1 of study had body weight loss coinciding with a period of low food consumption and during weeks 1-2 before pairing body weight gain was low. At this dose level, low body weight gain was observed throughout gestation and until Day 4 of lactation. Food consumption was also low during this time often attaining statistical significance. At 4500 ppm females had low body weight gain during the period prior to pairing and during Days 0-6 of gestation, for these animals food consumption was also low for Days 1-4 of lactation. Females receiving 1500 ppm did not have any periods of statistically low food or body weight performance.

 

Visual water consumption was notably high in animals receiving 15000 or 4500 ppm. 

 

Minor changes in haematology and biochemistry parameters did not demonstrate an adverse effect of treatment at the degree observed.

 

Organ weights adjusted for terminal body weight indicated high kidney and liver weights for males receiving 15000 ppm, and slightly low heart and ovary weights for females at 15000 ppm, these differences did not correlate with any histopathological changes.

 

The macroscopic examination performed on the males after 6 weeks of treatment and on the females on Day 7 of lactation revealed no test substance related lesions. Histopathology investigations identified a treatment related change in the thymus of an increased incidence and severity for involution/atrophy seen in all females receiving 15000 ppm. This change was not observed at lower dose levels.

 

The screen for reproductive performance indicated there was no effect of Parathymol P16029 on pre-coital interval, fertility or gestation length. Clinical signs of offspring did not indicate any reaction to maternal exposure at dose levels up to 15000 ppm.

 

The mean number of implantations and subsequent mean litter size was lower than Controls for females receiving 15000 ppm. Despite this there was no subsequent effect of treatment upon post-partum offspring survival or sex ratio to Day 7 of age at 15000 ppm. Mean numbers of implantations, litter size and sex ratio at 4500 or 1500 ppm were similar to Controls.

 

At 15000 ppm mean body weight of male and female offspring on Day 1 of age were lower than Control, and subsequent body weight gain to Day 7 of age, was also lower than Controls. Offspring body weight on Day 1 of age, and subsequent body weight gain to Day 7 of age, at 4500 or 1500 ppm was similar to Controls, and unaffected by treatment. According the authors, it is likely that the low body weight gain in offspring was associated with poor maternal body weight performance, low food consumption and the resultant maternal stress. 

 

Changes detected at necropsy on offspring killed or dying at or before scheduled termination were part of the normal spectrum expected in this strain of animal, at these ages and at this laboratory.

Under the test conditions, the NOAEL of Parathymol P16029 to male and females adult rats for at least 5 weeks was 15000 ppm (equivalent to 1000 mg/kg bw/d). Low implantation site counts impacting subsequent litter size was observed at 15000 ppm. However, the significantly reduced food consumption and reduced bodyweight gain in the females receiving 15000 ppm is considered to be an important factor for the reduced implantations/litter size as described in the literature (Terry et al., 2005) and this interpretation is strengthened by the observation that females receiving 15000 ppm showed involution/atrophy of the thymus which was considered to be indicative of a stress response secondary to the reduced food intake. This was an isolated finding as there was no effect of treatment on mating performance or fertility, the survival of the offspring and no treatment related macroscopic or microscopic changes were detected in the male and female reproductive systems. Therefore, the low implantation counts and litter size at 15000 ppm were considered to be associated with the reduced food consumption and not to reflect an intrinsic, specific property of Parathymol” (UN GHS for labelling of chemicals).