4-trans-propenylveratrole

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 19 to June 25, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline 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 421 (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 February 05-07, 2014/ signed on April 24, 2014)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-trans-propenylveratrole

EC Number:

228-958-7

EC Name:

4-trans-propenylveratrole

Cas Number:

6379-72-2

Molecular formula:

C11H14O2

IUPAC Name:

(E)-1,2-Dimethoxy-4-prop-1-en-1-ylbenzene

Test material form:

liquid

Details on test material:

- Physical state: Pale yellow, oily liquid
- Storage condition of test material: Room 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) Ltd.
- Age at study initiation: 71 to 78 days
- Weight at study initiation: 331-393 g for males; 225-276 g for females
- Housing: Pre-pairing - 5/sex/cage; Pairing – 1 male:1 female per cage; Males after mating – 5 males/cage; Gestation – 1 female /cage; Lactation – 1 female/cage (+ litter); Solid (polycarbonate) bottom cages were used during the acclimatisation, gestation, littering and lactation periods; Grid bottomed cages were used during pairing.
- Diet: SDS VRF1 Certified powdered diet, ad libitum
- Water: Potable water taken from the public supply, 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 light

IN-LIFE DATES: From January 19 to June 25, 2015

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: SDS VRF1 certified diet

Details on exposure:

DIET PREPARATION
- Method of preparation: On each occasion of the preparation of the premix, the required amount of test substance was added to an equal amount of plain diet and stirred until visibly homogenous. This doubling up process was repeated until half of the final weight of premix was achieved. 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 200 cycles to ensure the test substance was dispersed in the diet.
Aliquots of the premix were then diluted with further quantities of plain diet to produce the required dietary concentrations. Each batch of treated diet was mixed for a further 200 cycles in a Turbula mixer.
- Frequency of preparation: Weekly
- Storage of formulation: Ambient temperature.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: After a minimum of two weeks of treatment, males and females were paired on a one-to-one basis from within the same treatment group for a period of up to two weeks.
- Proof of pregnancy: Day on which evidence of mating was found (Ejected copulation plugs in cage tray and sperm in the vaginal smear) was designated Day 0 of gestation.
- After successful mating each pregnant female was caged: 1/cage
- Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 1000 and 20000 ppm were analysed to assess the stability and homogeneity of the test substance in the diet matrix.
Stability was confirmed as 22 days at ambient temperature (nominally 21 °C) and 22 days frozen (nominally -20 °C).

Achieved concentration: Samples of each formulation prepared for administration during treatment were analysed for achieved concentration of the test substance as follows: Day 1 (males and females); Week 4 (males); Day 6 of lactation (females)

Duration of treatment / exposure:

F0 animals:
Males - for a minimum of four weeks.
Females - for 15 days before pairing until Day 6 after birth of F1 generation.
F1 animals: No direct treatment
The F1 generation received no direct administration of the test substance; any exposure to the test substance or metabolites was through the mother to the offspring in utero and/or through the milk.

Frequency of treatment:

Continuously

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 500 ppm (nominal)

Doses / concentrations 2

Dose / conc.:

4 500 ppm (nominal)

Doses / concentrations 3

Dose / conc.:

15 000 ppm (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dietary concentrations used in this study (0, 1500, 4500 and 15000 ppm) were selected in conjunction with the Sponsor. The dose levels were selected based on the results of a range-finding study, Huntingdon Life Sciences Study No. NLA0017. In this study, dietary concentrations of 7500 or 15000 ppm were administered to rats for 14 days. The results from this study demonstrated that treatment at 15000 ppm was generally well tolerated, and did not result in any adverse toxicity. There were no deaths or adverse clinical signs at any dietary level but food consumption and body weight gain were reduced during the first week of treatment. During the second week of treatment body weight gains were similar to Controls and food consumption had improved, however there was still a reduction in food intake for females given 15000 ppm. Additionally there were no treatment related findings at macroscopic examination. Based on these findings, the high dose level for this reproductive/developmental toxicity screening study (NLA0018) was 15000 ppm (equivalent to 1000 mg/kg bw/day). Low and intermediate dose levels of 1500 ppm and 4500 ppm (equivalent to 100 and 300 mg/kg bw/day) were selected based on the previous study.
- Rationale for animal assignment: Randomly allocated on arrival. Before Day 1 of treatment, animals were weighed and tattooed. On Day 1 (before dosing) variations in body weight of animals were confirmed to be within ±20% of the mean for each sex.

Positive control:

None

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: A detailed physical examination was performed on each animal to monitor general health at the following time points:
F0 males: Once each week
F0 females: Once each week before pairing, on Days 0, 7, 14 and 20 after mating and on Days 1 and 7 of lactation.

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 males: Before dosing on the day that treatment commenced (Day 1), weekly thereafter and before necropsy.
F0 females: Before dosing on the day that treatment commenced (Day 1), weekly before pairing, on Days 0, 3, 7, 10, 14, 17 and 20 after mating, on Days 1, 4, and 7 of lactation and before necropsy.

FOOD CONSUMPTION: Yes
- The weight of food supplied, that remaining and an estimate of any spilled was recorded as follows:
F0 males: Weekly before pairing.
F0 females: Weekly before pairing, on Days 0-2, 3-6, 7-9, 10-13, 14-16 and 17-19 after mating and on Days 1-3 and 4-6 of lactation.
From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each phase.

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

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

Oestrous cyclicity (parental animals):

Dry smears: Daily smears were taken for 15 days before pairing, using cotton swabs moistened with saline. Smears were subsequently examined to establish the duration and regularity of the oestrous cycle.
Wet smears: After pairing with the male, daily smearing was continued using pipette lavage, until evidence of mating was observed.

Sperm parameters (parental animals):

For the assessment of the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.

Litter observations:

PARAMETERS EXAMINED
Clinical observations: Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1 to 7 of age.
Sex ratio of each litter: Recorded on Days 1, 4 and 7 of age.
Individual offspring body weights: F1 offspring - Days 1, 4 and 7 of age.

GROSS EXAMINATION OF DEAD PUPS: Yes

Postmortem examinations (parental animals):

SACRIFICE
All adult animals were killed by carbon dioxide asphyxiation.
Male animals: All surviving F0 males were killed during Week 5 of treatment.
Maternal animals:
- F0 females surviving until the end of the scheduled study period were killed on Day 7 of lactation.
- F0 females that failed to produce a viable litter were killed on Day 25 after mating.

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.
- The number of uterine implantation sites were checked after staining with ammonium sulphide (modification of the Salewski staining technique (Salewski 1964)), for females failing to produce a viable litter only.

ORGAN WEIGHTS
For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for adult animals killed at scheduled necropsy as listed in the table 7.8.1/1.

HISTOPATHOLOGY
Fixation: Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:
Testes Initially, in modified Davidson’s fluid.
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 adult animals of Groups 1 and 4 killed at scheduled termination.
Abnormalities only: All adult animals.
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:
Terminal sacrifice:
All adult animals in Groups 1 and 4: All specified in Table 7.8.1/1
All adult animals from all groups: Abnormalities

For the assessment of the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.
For the assessment of the ovaries a qualitative evaluation of one section from each ovary was made.

Postmortem examinations (offspring):

SACRIFICE
All offspring were killed by an intraperitoneal injection of sodium pentobarbitone.

GROSS NECROPSY
F1 generation offspring
- Offspring at scheduled termination: Examined externally; those offspring deemed normal were discarded without further macroscopic examination. Any externally abnormal offspring were examined internally and any abnormal tissues were retained.
- Premature deaths (before Day 7 of age): Missing offspring and those grossly autolysed or grossly cannibalised could not be examined. All other offspring dying before Day 7 of age were subject to fresh macroscopic examination, where possible (external and internal); this also included an assessment for the presence of milk in the stomach, where this was possible.
Abnormal tissues retained.

Statistics:

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

Reproductive indices:

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

Offspring viability 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 7 after littering / Number of live offspring on Day 1 after littering) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

adverse effects on food consumption and weight gain, which were not fully alleviated in the recovery period

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

adverse effects on food consumption and weight gain, which were not fully alleviated in the recovery period

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

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

Description (incidence and severity):

- One control female was found to not be pregnant, all other females were pregnant.
- The number of implantations was slightly low for females receiving 15000 ppm (p<0.05), and as a consequence of this, and a slightly low post implantation survival index, the litter size for this group was also slightly low (p<0.01). Live birth index and viability index were similar to that of the controls and there was no effect of treatment on sex ratio in these animals.
- There were no treatment related findings on the litter parameters in females given 1500 or 4500 ppm.

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- The appearance and behaviour of the animals was unaffected by treatment and there were no premature deaths.

BODY WEIGHT (PARENTAL ANIMALS)
- Bodyweight loss was observed for males and females given 15000 ppm during the first week of treatment (-4.5% and -4.8% when comparing Day 1 with Day 8 absolute body weights, statistics on change period is p<0.001). Additionally, bodyweight gains were reduced for males (-15% compared to control, p<0.001) and weight stasis was recorded in females receiving 4500 ppm.
- During the second week of treatment male bodyweight gains were similar or slightly higher than the controls for all treated Groups. In addition, during the second week of treatment, body weight gains for females at 15000 ppm were also above that of the controls (+57% compared to control, p<0.001). The overall (Week 1 to 2) bodyweight gains were low for females receiving 4500 ppm (-55% compared to control, p<0.001), with a small loss observed in females receiving 15000 ppm (-105% compared to control, p<0.001). The overall bodyweight gain for males (Week 1 to 4) given 15000 ppm were also low when compared with the controls.
- From Day 10 of gestation and throughout lactation bodyweight gains were significantly low for females given 15000 ppm (-26 to -37% during gestation and -61% during lactation when compared with controls).

FOOD CONSUMPTION (PARENTAL ANIMALS)
- Food consumption during Week 1 was markedly low at 15000 ppm in males and females (-42 % and -46 % compared to Control, respectively; p < 0.01) and slightly low at 4500 ppm in females (-17 %, compared to Control; p < 0.05). During Week 2 of treatment there was an improvement in food intake for males, as all Groups were similar to controls. For females, however, food consumption remained slightly low at 4500 or 15000 ppm (-8.5 % for both groups compared to Control; p < 0.05).
- During gestation and lactation, food consumption remained low for females receiving 15000 ppm (up to – 26% and -33% compared to Control, respectively; p < 0.01, except during Days 14-16 where p < 0.05), and was slightly low during Days 0-2 of gestation for females receiving 4500 ppm (-13 % compared to Control; p < 0.05).

WATER CONSUMPTION (PARENTAL ANIMALS)
- Visual water consumption assessment revealed that at 15000 ppm on Days 4 to 6 of treatment consumed less water than the controls. During the second week of treatment, and during gestation and lactation, visual water consumption was similar to that of the controls.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
- Mean achieved doses before pairing were 94, 272 or 769 mg/kg bw/day respectively for males receiving 1500, 4500 or 15000 ppm and 103, 289 or 826 mg/kg bw/day for females. During gestation, mean achieved doses were 113, 329 or 1008 mg/kg bw/day respectively for females receiving 1500, 4500 or 15000 ppm. During lactation, due to increased physiological demand, the mean achieved doses were higher at 207, 601 or 1793 mg/kg bw/day respectively for females receiving 1500, 4500 or 15000 ppm.

REPRODUCTIVE FUNCTION
Oestrous cycles, mating performance, gestation length and index:
- A higher proportion (60% which was outside historical control range (0% to 30%), p<0.01) of females receiving 15000 ppm showed regular 4/5 day oestrus cycles (and thus a lower proportion showed a 4 day cycle), compared with controls (10%).
- There was no effect of the test material on pre-coital interval, mating performance, fertility, gestation length or gestation index.

ORGAN WEIGHTS (PARENTAL ANIMALS)
- There was no effect of treatment on organ weights.

GROSS PATHOLOGY (PARENTAL ANIMALS)
- The macroscopic examination performed after 4 weeks of treatment revealed no test substance related lesions.

HISTOPATHOLOGY (PARENTAL ANIMALS)
- Tissues from group 1 and group 4 male and female animals were examined microscopically. No treatment-related effects were identified following administration of test item in any of the tissues examined. All microscopic changes were consistent with the common background lesions seen in this species at these laboratories.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

(Screening for fertility and development (foetal and pup growth survival until day 7))

Effect level:

4 500 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

2

Key result

Dose descriptor:

NOAEL

Remarks:

(Screening for fertility)

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

food consumption and compound intake

3

Dose descriptor:

NOAEL

Remarks:

(Screening for fertility)

Effect level:

>= 289 - <= 601 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

other: NOAEL pre-pairing = 289 mg/kg bw/day; NOAEL gestation = 329 mg/kg bw/day; NOAEL lactation = 601 mg/kg bw/day

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- There was an increased incidence of pups with cold to touch and/or little milk in stomach in male and female offspring from F0 females receiving 15000 ppm.

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 15000 ppm, male and female offspring body weights on Day 1 of age and growth of male and female pups between Days 1 and 7 of age were significantly low, when compared with the controls (body weights were 7-9% lower on Day 1; not statistically significant and growth was ca 50% of Controls; p<0.01)

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- Macroscopic examination of the offspring revealed an increased incidence of no milk in stomach in male and female offspring killed before scheduled termination from F0 females receiving 15000 ppm.
- Among offspring examined on Day 7 of age, 23 pups in 2 litters in the 15000 ppm group had thin build; pups in one of these litters also had no milk in stomach, while pups in the other litter were small.

Histopathological findings:

no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

(Screening for fertility and development (foetal and pup growth survival until day 7))

Generation:

F1

Effect level:

4 500 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to other toxic effects:

not specified

Dose response relationship:

yes

Relevant for humans:

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, method accuracy and precision.

The homogeneity was confirmed for test item in SDS VRF1 certified diet formulations at nominal concentrations of 1000 ppm and 20000 ppm. Stability was confirmed at ambient temperature and frozen storage for up to 22 days. Standard stability was confirmed for up to 6 days when refrigerated.

The mean concentrations of test item in test formulations analysed for the study were within +10%/-15% of nominal concentrations, confirming accurate formulation. With the exception of Day 1 Group 2, difference from mean values remained within 5%, confirming precise analysis.

 

Micronucleus analysis

No statistically significant increases in the frequency of micronucleated polychromatic erythrocytes and no statistically significant decreases in the proportion of polychromatic erythrocytes were observed in male or females at any dose level, compared to vehicle control values.

 

Applicant's summary and conclusion

Conclusions:

Under the test conditions, the NOAEL for reproductive and developmental toxicity was 4500 ppm (equivalent to daily intakes between 272 and 601 mg/kg bw/day depending on the sex, age and weight of the animals) on the basis that the animals of the 15000 ppm dose group suffered adverse effects on food consumption and weight gain, which were not fully alleviated in the recovery period.

Executive summary:

In a Reproduction / Developmental Toxicity Screening Test conducted according to OECD Guideline 421 and in compliance with GLP, test item was administered to groups of Crl:CD(SD) rats (10/sex/dose) at dietary concentrations of 1500, 4500, or 15000 ppm by dietary administration. A similarly constituted Control group received the vehicle, SDS VRF1 certified diet. Males were treated for a minimum of four consecutive weeks, including two weeks prior to pairing. Females were treated for two weeks prior to pairing, throughout mating and gestation and until Day 7 of lactation. The F1 generation received no direct administration of the test substance; any exposure was in utero or via the milk. During the study, clinical condition, body weight, food consumption, organ weights, macroscopic and microscopic pathology investigations were undertaken in all adults and a micronucleus test was included in the study. Pre‑coital interval, gestation length and index and mating performance (males and females) and fertility were assessed and parturition observations were performed for F0 females. The clinical condition, litter size and survival, sex ratio and body weight of all offspring were assessed. 

The accuracy of formulation and the accuracy of analysis with the exception of Day 1 Group 2 were confirmed and procedural recovery values remained within validated limits, confirming the continued accuracy of the method.

There was no evidence that test item caused an increase in the frequency of micronucleated polychromatic erythrocytes, or that it caused bone marrow cell toxicity, in male or female rats.

Mean achieved doses before pairing were 94, 272 or 769 mg/kg bw/day respectively for males receiving 1500, 4500 or 15000 ppm and 103, 289 or 826 mg/kg bw/day for females. During gestation, mean achieved doses were 113, 329 or 1008 mg/kg bw/day respectively for females receiving 1500, 4500 or 15000 ppm. During lactation, due to increased physiological demand, the mean achieved doses were higher at 207, 601 or 1793 mg/kg bw/day respectively for females receiving 1500, 4500 or 15000 ppm. 

There was no effect on the appearance and behaviour of the F0 males and females and no premature deaths.

Food consumption was markedly low during Week 1 of treatment at 15000 ppm, but there was an improvement in males during Week 2. For females however, food intake remained low during gestation and lactation. At 4500 ppm, mean food consumption was slightly low during Days 0 to 2 of gestation. Bodyweight loss was recorded during the first week of treatment for both sexes given 15000 ppm, and low weight gain was recorded for females receiving 15000 ppm from Day 10 of gestation and throughout lactation.

 

Oestrous cycle assessment revealed that a higher proportion of females receiving 15000 ppm showed regular 4/5 day oestrus cycles compared with the controls. There was however, no effect on pre-coital interval, mating performance, fertility, and gestation length or gestation index.

There was no clear effect on organ weights and no treatment-related test substance related lesions during macroscopic or microscopic examination.

The number of implantations, post-implantation survival index, and litter size was low for females receiving 15000 ppm. There was, however, no effect of treatment on live birth index, viability index or sex ratio.

Offspring clinical signs revealed an increased incidence of pups with, cold to touch and/or little milk in the stomach at 15000 ppm. In addition, offspring body weights on Day 1 of age were slightly low and the growth of the offspring between Days 1 and 7 of age was markedly low. Macroscopic examination of the offspring terminated before Day 7 of lactation revealed an increased incidence of no milk in the stomach for offspring from F0 females given 15000 ppm and thin build was apparent among offspring in 2 litters at Day 7 of age.

It is concluded that administration of the substance to rats by dietary administration over a period of at least five weeks at levels of 1500, 4500 or 15000 ppm was generally well tolerated by the males and females before pairing. Bodyweight gain was reduced when compared with the controls

in females given 15000 ppm throughout the treatment period, but especially after Day 10 of gestation, and the associated food intake was also significantly reduced in these animals. At 15000 ppm, the mean number of implantations, the mean post implantation survival index and mean total and live litter sizes were slightly but significantly low, and the growth of the offspring was markedly low. The cause of the reduced food consumption and body weight gain in the dams, and the cold to touch and/or little milk in the stomach, observed in the offspring, and reduced growth of the offspring, is not certain. However, in the absence of significant effects during macroscopic examination and histopathology it is considered likely

to reflect an effect mediated through the reduced food consumption of the dam during lactation. The reduced food consumption was probably a consequence of an adverse impact on the palatability of the feed by the test substance, which is a fragrance material.

Under the test conditions, the NOAEL for reproductive and developmental toxicity was 4500 ppm (equivalent to daily intakes between 272 and 601 mg/kg bw/day depending on the sex, age and weight of the animals) on the basis that the animals of the 15000 ppm dose group suffered adverse effects on food consumption and weight gain, which were not fully alleviated in the recovery period.