Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
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:
10th June 2011 and 20 September 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the studywas conducted under GLP conditions.
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS

- Source:
Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK

- Age at study initiation:
Approx twelve weeks old

- Weight at study initiation:
(P) Males: 315 to 371g; Females: 191 to 225g

- Fasting period before study:
Not applicable.

- Housing:
Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the mating phase, the animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation, in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.

- Use of restrainers for preventing ingestion (if dermal):
no

- Diet (e.g. ad libitum):
The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK) was used throughout the study period. Environmental enrichment was provided in the form of wooden chew blocks (Datesand Ltd., Cheshire, UK).

- Water (e.g. ad libitum):
Mains drinking water was supplied from polycarbonate bottles attached to the cage ad libitum.

- Acclimation period:
The animals were acclimatised for 12 days, during which time there health status was assessed.

ENVIRONMENTAL CONDITIONS

- Temperature (°C):
Temperature was set to 21°C+/-2°C

- Humidity (%):
55 +/- 15%

- Air changes (per hr):
The rate of air exchange was at least 15 air changes per hour.

- Photoperiod (hrs dark / hrs light):
The low intensity flourescent lighting was controlled to give twelve hours continuous light and twelve hours darkness

IN-LIFE DATES: From:
Day 1 To: Day 46
Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
other: Not applicable
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

For the purpose of this study the test item was melted at 70ºC and then prepared at the appropriate concentrations as a solution in Arachis oil BP. The stability and homogeneity of the test item formulations were previously determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services (Harlan Laboratories Ltd. Project Number: 41101419). Results from the previous study showed the formulations to be stable for at least twenty one days. Formulations were therefore prepared twice monthly and stored at approximately 4ºC in the dark.
Samples of test item formulations were taken and analysed for concentration of 2,6-Bis(1,1-dimethylethyl)-4-(arylalkylidene)alicycli-2,5-dien-1-one at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within ± 5% of the nominal concentration.

DIET PREPARATION

- Rate of preparation of diet (frequency):
Not applicable

- Mixing appropriate amounts with (Type of food):
Not applicable

- Storage temperature of food:
Not applicable

VEHICLE
- Amount of vehicle (if gavage): 4 ml/kg
Details on mating procedure:

- M/F ratio per cage:
One male: One Female per cage

- Length of cohabitation:
14 days

- Proof of pregnancy:
Cage tray liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded, referred to as day 0 of pregnancy


- After successful mating each pregnant female was caged:
Mated females were housed individually, during the period of gestation and lactation.

- Any other deviations from standard protocol:
No
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
See attached chemistry report
Duration of treatment / exposure:
42 days for males and up to 46 Days for females
Frequency of treatment:
The test material was administered daily by gavage using a stainless steel cannula attached to disposable plastic syringe.
Details on study schedule:
Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable)

On Day 15, all animals were paired on a 1 male:1 female basis within each dose group for a maximum of fourteen days.

Following evidence of mating the males were returned to their original cages and females were transferred to individual cages.

Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Evaluation of each litter size, litter weight, mean pup weight, clinical observations and landmark developmental signs were also performed during this period.

At Day 5 post partum, all females and offspring were killed and examined macroscopically.

Remarks:
Doses / Concentrations:
0 mg/kg bw/day (0 mg/ml)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
30 mg/kg bw/day AI (7.5 mg/ml AI)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg bw/day AI (25 mg/ml AI)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
250 mg/kg bw/day AI (62.5 mg/ml AL)
Basis:
actual ingested
No. of animals per sex per dose:
Control-10 males and 10 females 0 mg/kg bw/day
Low- 10 males and 10 females 30mg/kg bw/day
Intermediate-10 males and 10 females 100mg/kg bw/day
High-10 males and 10 females 250mg/kg bw/day
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were chosen based on previous toxicity data.

-Rationale for animal assignment (if not random):
Random

- Other:
The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test material, and the results of the study are beleived to be of value in screening potential adverse effects on reproduction.
Positive control:
Not applicable
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Observed immediately before and up to 30 minutes after dosing, and one and five hours after dosing, during the working week. Animals were observed immeadiately before and up to 30 minutes after dosing, and one hour after dosing at weekends and on public holidays (except for females during parturition where applicable).

DETAILED CLINICAL OBSERVATIONS:
- Time schedule:
Observed immediately before and up to 30 minutes after dosing, and one and five hours after dosing, during the working week. Animals were observed immeadiately before and up to 30 minutes after dosing, and one hour after dosing at weekends and on public holidays (except for females during parturition where applicable).

BODY WEIGHT:
Yes
- Time schedule for examinations:
Individual bodyweights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident. Bodyweights were then recorded for females on Days 0,7,14 and 20 post coitum, and on Days 1, 4 and 5 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):

- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

During the maturation period, weekly food consumption was recorded for each cage of treated adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering Days 0-7,7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 5 post partum.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations:
Water intake was measured daily throughout the study (with the exception of the mating phase).

OTHER:
Oestrous cyclicity (parental animals):
No Data
Sperm parameters (parental animals):
Parameters examined in male parental generations:
testis weight, epididymis weight,
Litter observations:
STANDARDISATION OF LITTERS

- Performed on day 4 postpartum: No

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, other: Results of the surface righting assessments on Day 1 post partum, Clinical condition of offspring from birth to Day 5 post partum, Inviduals litter and offspring weights on Day 1 and 4 post partum.


GROSS EXAMINATION OF DEAD PUPS:
yes, full external and internal examination.
Postmortem examinations (parental animals):
SACRIFICE

- Male animals: All surviving males were teminated on Day 43.

- Maternal animals: All treated females and offspring were killed on day 5 post partum

GROSS NECROPSY

- Adult males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of Sodium pentobarbitone on Day 5 post partum.

All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

In addition, the corpora lutea of all ovaries and uterine implantation sites from pregnant females were counted at necropsy. The procedure for counting implantation sites was enhanced (where necesary) by staining the uteri with a 0.5% ammonium polysulphide solution.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in the attached histopathology report were prepared for microscopic examination. The testes and epididymides were weighed.
Postmortem examinations (offspring):
SACRIFICE
Day 5 post partum


GROSS NECROPSY
All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

HISTOPATHOLOGY / ORGAN WEIGTHS
No data
Statistics:
The following parameters were subjected to statistical analysis:
Body weight and body weight change
Food consumption for females during gestation and lactation
Pre-coital interval and gestation length
Litter size and litter weights
Sex ratio
Corpora lutea and implantation sites
Implantation losses and viability indices
Offspring body weight and body weight change
Offspring surface righting
Adult absolute and body weight-relative organ weights

For full details please see "Statistical Proceedures used" in Any other information on materials and methods incl. tables section
Reproductive indices:
Mating performance and Fertlity
The following parameters were calculated from the individual data during the mating period of the parental generation.

Pre coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

Fertility Indices

For each group the following were calculated:

Mating Index (%) = Number of animals mated/Number of animals paired x 100
Pregnancy Index(%) = Number of pregnant females/Number of animals mated
Offspring viability indices:
Live Birth Index(%) = Number of offspring alive on Day 1/ Number of offspring born x 100
Viability Index1 (%) = Number of offspring alive on Day 4/ Number of offspring alive on Day 1 x 100
Clinical signs:
effects observed, treatment-related
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
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
effects observed, treatment-related
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
effects observed, treatment-related
Reproductive performance:
effects observed, treatment-related
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)

One male treated with 250 mg/kg bw/day was found dead on Day 9. The exact cause of death of this animal could not be established microscopically
There were no further unscheduled deaths.

A summary incidence of clinical observations is given in attached Table 2. Individual clinical observations are presented in attached Appendix 1.
Increased salivation was evident in animals of either sex, with increased frequency from all treatment groups throughout the treatment period. One male treated with 250 mg/kg bw/day showed an isolated incident of orange fur staining on Day 2.
The male treated with 250 mg/kg bw/day that was found dead on Day 9 had orange coloured diarrhoea on Day 4 only.
One female treated with 30 mg/kg bw/day also had a mass beneath the right forelimb from Day 14 onwards. This was confirmed during microscopic evaluation as an abscess and was considered not to be of toxicological significance.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)

Group mean body weights, body weight changes and standard deviations are given in attached Table 3 and Table 4 (statistically significant differences are indicated). Group mean body weights are presented graphically in attached Figure 1 and Figure 2. Individual data are given in attached Appendix 2 to Appendix 3.

-Maturation/Post pairing males
Males treated with 250 mg/kg bw/day showed actual body weight losses during the first week of treatment resulting in a statistically significant (P<0.01) reduction in body weight gain. A reduction in body weight gain was also evident in these animals during Weeks 3 and 4 however statistical significance (P<0.01) was only achieved during Week 4.
Males treated with 100 mg/kg bw/day showed a statistically significant (P<0.05) reduction in body weight gain during the first week of treatment.

-Gestation
No adverse effect on bodyweight development was detected was detected throughout gestation.

-Lactation
No adverse effect on bodyweight development was detected throughout lactation.

Group mean food consumptions are given in attached Table 5 and are presented graphically in attached Figure 3 and Figure 4. Individual values for females during gestation and lactation are presented in attached Appendix 4. Food efficiency for males and for females during the pre-mating phase is given in attached Table 6.

-Maturation/ Post pairing males
Males treated with 250 mg/kg bw/day showed a reduction in food consumption during Week 1 and Week 5. A reduction in food efficiency was also evident in these males during the first week of treatment.

-Gestation
No adverse effect on dietary intake or food efficiency was detected throughout gestation.

-Lactation
No adverse effect on dietary intake or food efficiency was detected throughout lactation.
TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
No data

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No data

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No data

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
A summary of adult performance is presented in attached Table 1. Group values and summary incidence for mating performance are presented in attached Table 8. Individual data are given in attached Appendix 6.

There were no treatment-related effects on mating performance. With the exception of one 250 mg/kg bw/day pair, which did not show confirmation of mating, the remaining paired animals mated within the first four days of pairing.

Group values for fertility, litter data and implantation losses are given in attached Tables 8, 9 and 10. Individual data are given in attached Appendices 6 to 8.
No treatment-related effects were detected on fertility for treated animals when compared to controls.

ORGAN WEIGHTS (PARENTAL ANIMALS)

Group mean absolute and body weight-relative organ weights and standard deviations for test and control group animals are presented in attached Table 14 (statistically significant differences are indicated). Individual data are given in attached Appendix 12 and Appendix 13.
Females treated with 250 and 100 mg/kg bw/day showed a statistically significant (P<0.01 and P<0.05 respectively) increase in liver weight both absolute and relative to body weight. No such effects were detected in males treated 250 or 100 mg/kg bw/day or animals of either sex treated with 30 mg/kg bw/day.
No treatment related effects were detected in the reproductive organs weighed in males from any treatment group.


GROSS PATHOLOGY (PARENTAL ANIMALS)

A summary incidence of necropsy findings for adults is given in attached Table 16. Individual data are given in attached Appendices 15.

The male treated with 250 mg/kg bw/day that was found dead on Day 9 did not show any macroscopic abnormalities.
No toxicologically significant macroscopic abnormalities were detected in terminal kill animals of either sex treated with 250 or 100 mg/kg bw/day or males treated with 30 mg/kg bw/day.
One female treated with 30 mg/kg bw/day had a mass in the right thoracic region at necropsy. This was confirmed during microscopic evaluation as an abscess and was considered not to be of toxicological significance


HISTOPATHOLOGY (PARENTAL ANIMALS)

A complete histopathology report is attached.

The following treatment related microscopic findings were detected:
Liver: minimal centrilobular hypertrophy was evident in animals of either sex treated with 250 mg/kg bw/day and in males treated with 100 mg/kg bw/day. The incidence and severity of glycogen deposit of hepatocytes were also decreased as a consequence of the centrilobular hypertrophy in males treated with 250 mg/kg bw/day.

OTHER FINDINGS (PARENTAL ANIMALS)

Water consumption: Group mean water consumptions are given in attached Table 7. Individual and group mean water consumptions for females following mating and lactation are presented in attached Appendix 5.
There were no adverse effects detected in water consumption for treated animals when compared to control animals.

Gestation Length:

A summary incidence of gestation lengths is given in attached Table 8. Individual lengths are given in attached Appendix 6.
No treatment-related effects were detected in the length of gestation for treated females when compared to controls. All animals showed gestation lengths between 22½ to 23½ days.


Dose descriptor:
NOAEL
Remarks:
Systemic toxicity
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The microscopic liver changes detected in 100 mg/kg bw/day males and the increased liver weights in females at 100 mg/kg bw/day in isolation were considered to be adaptive in nature and not to represent an adverse effect of treatment.
Dose descriptor:
NOEL
Remarks:
Reproductive toxicity
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related effects were detected in the reproductive parameters examined
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
VIABILITY (OFFSPRING)

Group mean corpora lutea and implantation counts, litter size, implantation losses, survival indices and sex ratio are given in attached Tables 9 to 11. Individual data are given in attached Appendices 7 to 9.

No significant differences were detected for corpora lutea, implantation counts, litter size or litter viability for treated animals when compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences. There were no intergroup differences in sex ratio (percentage male offspring) for litters from treated groups compared to controls.


CLINICAL SIGNS (OFFSPRING)

A summary incidence of clinical signs and surface righting reflex are given in attached Tables 12 and 13. Individual observations are given in attached Appendices 10 ans 11.

No obvious clinical signs of toxicity were detected for offspring from treated females when compared to controls. The incidental clinical signs detected throughout the control and treated groups, consisting of small size, offspring found dead or missing, bruising, no milk in stomach, cold, weak, swollen abdomen and pale were considered to be low incidence findings observed in offspring in studies of this type, and were unrelated to test item toxicity. The percentage of offspring who successfully showed surface righting reflex on Day 1 was similarly unaffected by maternal exposure.

BODY WEIGHT (OFFSPRING)

Group mean values for total litter weights, offspring body weights and body weight changes are given in attached Table 9. Individual values and observations are given in attached Appendix 7.

There were no significant differences in litter or offspring weights. Statistical analysis of the data did not reveal any significant intergroup differences.


SEXUAL MATURATION (OFFSPRING)
No data

ORGAN WEIGHTS (OFFSPRING)

No data

GROSS PATHOLOGY (OFFSPRING)

A summary incidence of necropsy findings for offspring is given in attached Table 15. Individual data are given in attached Appendix 14.

No treatment-related macroscopic abnormalities were detected for interim death or terminal kill offspring. The incidental findings observed were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test item.

HISTOPATHOLOGY (OFFSPRING)

No data

OTHER FINDINGS (OFFSPRING)
Reproductive effects observed:
not specified
Conclusions:
The oral administration of 2,6-Bis(1,1-dimethylethyl)-4-(arylalkylidene)alicycli-2,5-dien-1-one to rats by gavage, at dose levels of 30, 100 and 250 mg/kg bw/day, resulted in treatment related effects detected in animals of either sex treated with 250 mg/kg bw/day.
The microscopic liver changes detected in 100 mg/kg bw/day males and the increased liver weights in females at 100 mg/kg bw/day in isolation were considered to be adaptive in nature and not to represent an adverse effect of treatment. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 100 mg/kg bw/day.
No treatment related effects were detected in the reproductive parameters examined therefore the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 250 mg/kg bw/day.
Executive summary:

Introduction.

The study was performed to screen for potential adverse effects of the test item on reproduction including offspring development and provides an initial hazard assessment for effect on reproduction. The study was designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 27 July 1995).

This study was also designed to meet the requirements of Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

The dose levels were chosen based on the results of previous toxicity work (Harlan Laboratories Ltd., Project Number 41101419).

Methods.

The test item was administered by gavage to three groups, each of ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week maturation phase, pairing, gestation and early lactation for females), at dose levels of 30, 100 and 250 mg/kg bw/day (adjusting for 12.3% solvent content). A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP).

Clinical signs, body weight change, dietary intake and water consumption were monitored during the study. 

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Survivingadult males were terminated on Day 43, followed by the termination of all females and offspring on Day 5 post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of liver and reproductive tissues was performed.

Results.

Adult Responses:

Mortality.One male treated with 250 mg/kg bw/day was found dead on Day 9. There were no further unscheduled deaths.

Clinical Observations. Episodes of increased salivation were evident in animals of either sex from all treatment groups throughout the treatment period. The male treated with 250 mg/kg bw/day that was found dead on Day 9 had orange coloured diarrhoea on Day 4. 

Body Weight. Males treated with 250 mg/kg bw/day showed actual body weight losses during the first week of treatment. A reduction in body weight gain was also evident in these animals during Weeks 3 and 4. Males treated with 100 mg/kg bw/day showed a reduction in body weight gain during the first week of treatment. No such effects were detected in females treated with 250 or 100 mg/kg bw/day or animals of either sex treated with 30 mg/kg bw/day.

Food Consumption and Food Efficiency.Males treated with 250 mg/kg bw/day showed a reduction in food consumption during Week 1 and Week 5. A reduction in food efficiency was also evident in these males during the first week of treatment. 

No toxicologically significant effects were detected in females treated with 250 mg/kg bw/day or animals of either sex treated with 100 or 30 mg/kg bw/day.

Water Consumption. No adverse effect on water consumption was detected.

Reproductive Screening:

Mating. There were no treatment-related effects on mating for treated animals.

Fertility. There were no treatment-related effects on conception rates for treated animals.

Gestation Lengths. There were no differences in gestation lengths. The distribution for treated females was comparable to controls.

Litter Responses:

Offspring Litter Size, Sex Ratio and Viability. Of the litters born, litter size at birth and subsequently on Day 1 and 4post partumwere comparable to controls. There were no intergroup differences in sex ratio.

Offspring Growth and Development. Offspring body weight gain and litter weights at birth and subsequently on Day 1 and 4post partumwere comparable to controls. No effect on surface righting reflex was detected.

Offspring Observations. No clinically observable signs of toxicity were detected for offspring from all treatment groups.

Pathology:

Necropsy. No toxicologically significant macroscopic abnormalities were detected in animals of either sex treated with 250, 100 or 30 mg/kg bw/day.

Organ Weights.Females treated with 250 and 100 mg/kg bw/day showed an increase in liver weight both absolute and relative to body weight.

No such effects were detected in males treated with 250 or 100 mg/kg bw/day or animals of either sex treated with 30 mg/kg bw/day.

No treatment related effects were detected in the reproductive organs weighed in males from any treatment group.

Histopathology.The following treatment related microscopic findings were detected:

Liver:minimal centrilobular hypertrophy was evident in animals of either sex treated with 250 mg/kg bw/day and in males treated with 100 mg/kg bw/day. The incidence and severity of glycogen deposit of hepatocytes were also decreased as a consequence of the centrilobular hypertrophy in males treated with 250 mg/kg bw/day.

Conclusion.The oral administration of 2,6-Bis(1,1-dimethylethyl)-4-(arylalkylidene)alicycli-2,5-dien-1-one to rats by gavage, at dose levels of 30, 100 and 250 mg/kg bw/day,resulted in treatment related effects detected in animals of either sex treated with 250 mg/kg bw/day.

The microscopic liver changes detected in 100 mg/kg bw/day males and the increased liver weights in females at 100 mg/kg bw/day in isolation were considered to be adaptive in nature and not to represent an adverse effect of treatment. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 100 mg/kg bw/day.

No treatment related effects were detected in the reproductive parameters examined therefore the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 250 mg/kg bw/day.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The oral administration of test substance to rats by gavage, at dose levels of 30, 100 and 250 mg/kg bw/day, resulted in treatment related effects detected in animals of either sex treated with 250 mg/kg bw/day.

 

The microscopic liver changes detected in 100 mg/kg bw/day males and the increased liver weights in females at 100 mg/kg bw/day in isolation were considered to be adaptive in nature and not to represent an adverse effect of treatment. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 100 mg/kg bw/day.

 

No treatment related effects were detected in the reproductive parameters examined therefore the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 250 mg/kg bw/day.


Short description of key information:
No treatment related effects were detected in the reproductive parameters examined therefore the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 250 mg/kg bw/day.

Justification for selection of Effect on fertility via oral route:
The NOAEL is derived from systemic toxicity observed in the parent during the OECD 421 study. Thoe NOEL for reproductive toxicity was 250 mg/kg bw/day.

Effects on developmental toxicity

Description of key information
No treatment related effects were detected in the reproductive parameters examined therefore the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 250 mg/kg bw/day.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 June 2011 to 20 September 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the studywas conducted under GLP conditions.
Qualifier:
according to
Guideline:
other: OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 27 July 1995).
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No 440/2008 of 30 May 2008 test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK

- Age at study initiation:
Approximately 12 weeks old

- Weight at study initiation:
315 to 371g (male); 191 to 225g (female)

- Fasting period before study:
Not applicable

- Housing:
Initially, all animals were housed in groups of five in solid floor polypropylene cages with
stainless steel mesh lids and softwood flake bedding (Harlan UK Ltd). During the mating
phase, animals were transferred to polypropylene grid floor cages suspended over trays
lined with absorbent paper on a one male: one female basis within each dose group.
Following evidence of successful mating, the males were returned to their original cages.
Mated females were housed individually during gestation and lactation, in solid floor
polypropylene cages with stainless steel mesh lids and softwood flakes.

- Use of restrainers for preventing ingestion (if dermal):
Not applicable

- Diet:
The animals were allowed free access to food. A pelleted diet Rodent 2018C
Teklad Global Certified Diet Harlan UK Ltd, Oxon, UK was used throughout the study
period. The diet was considered not to contain any contaminant at a level that might
have affected the purpose or integrity of the study.

- Water:
The animals were allowed free access to water. Mains drinking water was supplied from polycarbonate bottles attached to the cage. The drinking water was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

- Acclimation period:
For 12 days

ENVIRONMENTAL CONDITIONS

- Temperature (°C):
21 ± 2

- Humidity (%):
55 ± 15

- Air changes (per hr):
At least fifteen air changes per hour

- Photoperiod (hrs dark / hrs light):
12 hours continuous light and 12 hours darkness

IN-LIFE DATES:
Up to 42 consecutive days for males and up to 46 days for females
Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
other: Not applicable
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was melted at 70ºC and then prepared at the appropriate concentrations as a solution in Arachis oil BP. The stability and homogeneity of the test item formulations were previously determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services (Harlan Laboratories Ltd. Project Number: 41101419). Results from the previous study showed the formulations to be stable for at least twenty one days. Formulations were therefore prepared twice monthly and stored at approximately 4ºC in the dark. Samples of test item formulations were taken and analysed for concentration of 2,6-Bis(1,1-dimethylethyl)-4-(arylalkylidene)alicycli-2,5-dien-1-one at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The method used for analysis of formulations and the results obtained are given in the attached chemistry report. The results indicate that the prepared formulations were within ± 5% of the nominal concentration.

DIET PREPARATION
A pelleted diet Rodent 2018C Teklad Global Certified Diet from a reputable supplier please see full study report for details

- Rate of preparation of diet (frequency):
Not applicable

- Mixing appropriate amounts with (Type of food):
Not applicable

- Storage temperature of food:
No data

VEHICLE
Arachis oil

- Justification for use and choice of vehicle (if other than water):
Not applicable

- Concentration in vehicle:
62.5, 25 and 7.5 mg/ml AI

- Amount of vehicle (if gavage):
4 mg/kg

- batch no.:
SL0G1726A0

- Purity:

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:


see attached chemistry report
Details on mating procedure:
- M/F ratio per cage:
1/1 (Animals were paired on a 1 male: 1 female basis within each dose group)

- Length of cohabitation:
Up to 14 days

- Proof of pregnancy:
Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation)

- After successful mating each pregnant female was caged:
Mated females were housed individually during the period of gestation and lactation.

- Any other deviations from standard protocol:
Not applicable
Duration of treatment / exposure:
Up to 42 consecutive days for males and up to 46 Days for females
Frequency of treatment:
Daily
Duration of test:
Up to 42 consecutive days for males and up to 46 days for females
Remarks:
Doses / Concentrations:
30 mg/kg bw/day AI (7.5 mg/ml AI)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg bw/day AI (25 mg/ml AI)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
250 mg/kg bw/day AI (62.5 mg/ml AI)
Basis:
actual ingested
No. of animals per sex per dose:
10 animals per sex per dose (including control).
Control animals:
yes, concurrent vehicle
Details on study design:

- Dose selection rationale:
The dose levels were chosen based on the results of a previous range-finder study

- Rationale for animal assignment (if not random):
Random.

- Other:
Not applicable
Maternal examinations:
CAGE SIDE OBSERVATIONS:
Yes
- Time schedule:
Immediately before dosing, up to 30 minutes after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, up to 30 minutes after dosing, and one hour after dosing at weekends and public holidays (except for females during parturition where applicable).

DETAILED CLINICAL OBSERVATIONS:
Yes
- Time schedule:
Immediately before dosing, up to thirty minutes after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, up to 30 minutes after dosing, and one hour after dosing at weekends and public holidays (except for females during parturition where applicable).

BODY WEIGHT:
Yes
- Time schedule for examinations:
Individual bodyweights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident. Bodyweights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

FOOD CONSUMPTION:
Yes
- During the maturation period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum. Weekly food consumptions were performed weekly for each cage of adults throughout the study period.

FOOD EFFICIENCY:
Yes
- Food efficiency (the ratio of bodyweight change/dietary intake) was calculated retrospectively for males and females throughout the study period.

WATER CONSUMPTION: Yes
- Time schedule for examinations:Water intake was measured daily throughout the study (with the exception of the mating phase).

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice
Male animals: Adult surviving males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43.
Maternal animals: Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Any females that failed to achieve pregnancy or produce a litter were killed on or after Day 26 post coitum.


- Organs examined:
Gross pathology: For all females the uterus was examined for signs of implantation and the number of uterine implantations in each born was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 1% ammonium polysulphide solution. In addition, the corpora lutea of all ovaries from pregnant females were counted at necropsy. All adult animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Histopathology/organ weights:
The epididymides and testes were removed from terminal kill adult males dissected free from fat and weighed before fixation. The liver was removed from terminal kill adult animals dissected free from fat and weighed before fixation.


Samples of the following tissues were preserved from all animals from each dose group, in buffered 10% formalin, except where stated:
Coagulating gland, Pituitary, Epididymides, Prostate, Gross Lesions, Seminal vesicles, Liver, Testes, Ovaries, Uterus/Cervix, Mammary gland, Vagina


All tissues were despatched to the Test Site Harlan Laboratories Ltd., Zelgliweg 1,
CH-4452 Itingen, Switzerland for processing (Principal Investigator: K Weber). The tissues from control and 250 mg/kg bw/day dose group animals, livers from all animals, any animals dying during the study, and any animals which failed to mate or did not achieve a pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with Haematoxylin and Eosin for subsequent microscopic examination. In addition, sections of testes and epididymides from all control and 250 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.


OTHER:

PREGNANCY AND PARTURITION
Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and around the period of expected parturition. Observations were carried out at approximately 0830 and 1230 hours at weekends and public holidays. The following was recorded for each female:
i) Date of mating
ii) Date and time of observed start of parturition
iii) Date and time of observed completion of parturition
iv) Duration of gestation

Estrous cyclicity (Parental)
A vaginal smear was prepared for each female and the stage of the oestrous cycle was recorded.


Ovaries and uterine content:
The ovaries and uterine content was examined after termination:
Yes
Examinations included:
- Gravid uterus weight:
No

- Number of corpora lutea:
Yes

- Number of implantations:
Yes

- Number of early resorptions:
No

- Number of late resorptions:
No

- Other:

Fetal examinations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum:
No

PARAMETERS EXAMINED
The following parameters were examined in offspring: Number of offspring born, number and sex of offspring alive recorded daily and reported on Day 1 and 4 post partum, clinical condition of offspring from birth to Day 5 post partum, individual offspring and litter weights on Day 1 and 4 post partum, physical Development and pathology.

GROSS EXAMINATION OF DEAD PUPS:
Dying and dead offspring during the study were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

POST-MORTEM EXAMINATION
Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone. All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities wererecorded.

Statistics:
The following parameters were subjected to statistical analysis:
Body weight and body weight change
Food consumption for females during gestation and lactation
Pre-coital interval and gestation length
Litter size and litter weights
Sex ratio
Corpora lutea and implantation sites
Implantation losses and viability indices
Offspring body weight and body weight change
Offspring surface righting
Adult absolute and body weight-relative organ weights

For full ststistical procedures used please see any other information on materials and method inc. tables
Indices:
Not applicable.
Historical control data:
Not applicable.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
(Clinical signs)
A summary incidence of clinical observations is given in attached Table 2. Individual clinical observations are presented in attached Appendix 1.

Increased salivation was evident in animals of either sex, with increased frequency from all treatment groups throughout the treatment period. One male treated with 250 mg/kg bw/day showed an isolated incident of orange fur staining on Day 2.
The male treated with 250 mg/kg bw/day that was found dead on Day 9 had orange coloured diarrhoea on Day 4 only.
One female treated with 30 mg/kg bw/day also had a mass beneath the right forelimb from Day 14 onwards. This was confirmed during microscopic evaluation as an abscess and was considered not to be of toxicological significance.

(Mortality)
One male treated with 250 mg/kg bw/day was found dead on Day 9. The exact cause of death of this animal could not be established microscopically. There were no further unscheduled deaths.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
(Body weight)
Group mean body weights, body weight changes and standard deviations are given in attached Table 3 and Table 4 (statistically significant differences are indicated). Group mean body weights are presented graphically in attached Figure 1 and Figure 2. Individual data are given in attached Appendix 2 to Appendix 3.

Males treated with 250 mg/kg bw/day showed actual body weight losses during the first week of treatment resulting in a statistically significant (P<0.01) reduction in body weight gain. A reduction in body weight gain was also evident in these animals during Weeks 3 and 4 however statistical significance (P<0.01) was only achieved during Week 4.
Males treated with 100 mg/kg bw/day showed a statistically significant (P<0.05) reduction in body weight gain during the first week of treatment.


(Food consumption)
Group mean food consumptions are given in attached Table 5 and are presented graphically in attached Figure 3 and Figure 4. Individual values for females during gestation and lactation are presented in attached Appendix 4.
Food efficiency for males and for females during the pre-mating phase is given in attached Table 6.

Males treated with 250 mg/kg bw/day showed a reduction in food consumption during Week 1 and Week 5. A reduction in food efficiency was also evident in these males during the first week of treatment.
No toxicologically significant effects were detected in females treated with 250 mg/kg bw/day or animals of either sex treated with 100 or 30 mg/kg bw/day.
Females treated with 100 mg/kg bw/day showed a statistically significant (P<0.05) reduction in food consumption between Days 1 and 4 of lactation. In the absence of a true dose related response the intergroup difference was considered not to be of toxicological importance.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
(Mating)
A summary of adult performance is presented in attached Table 1. Group values and summary incidence for mating performance are presented in attached Table 8. Individual data are given in attached Appendix 6.

There were no treatment-related effects on mating performance.
With the exception of one 250 mg/kg bw/day pair, which did not show confirmation of mating, the remaining paired animals mated within the first four days of pairing.


(Fertility)
A summary of adult performance is given in attached Table 1. Group values for fertility, litter data and implantation losses are given in attached Tables 8, 9 and 10. Individual data are given in attached Appendices 6 to 8.

No treatment-related effects were detected on fertility for treated animals when compared to controls.
One female treated with 250 mg/kg bw/day did not achieve pregnancy. In the absence of any histopathological correlates in the reproductive organs to elucidate the cause of the non-pregnancy in this female the intergroup difference was considered to be incidental and of no toxicological importance.
One female treated with 100 mg/kg bw/day showed evidence of corpora lutea and implantation sites but did not give birth to any live offspring. In the absence of any histopathological correlates in the reproductive organs in either the paired female or male partner, this was considered to be incidental and of no toxicological importance.


(gestation length)
A summary incidence of gestation lengths is given in attached Table 8. Individual lengths are given in attached Appendix 6.
No treatment-related effects were detected in the length of gestation for treated females when compared to controls. All animals showed gestation lengths between 22½ to 23½ days


ORGAN WEIGHTS (PARENTAL ANIMALS)
Group mean absolute and body weight-relative organ weights and standard deviations for test and control group animals are presented in attached Table 14 (statistically significant differences are indicated). Individual data are given in attached Appendix 12 and Appendix 13.

Females treated with 250 and 100 mg/kg bw/day showed a statistically significant (P<0.01 and P<0.05 respectively) increase in liver weight both absolute and relative to body weight.
No such effects were detected in males treated 250 or 100 mg/kg bw/day or animals of either sex treated with 30 mg/kg bw/day.
No treatment related effects were detected in the reproductive organs weighed in males from any treatment group.


GROSS PATHOLOGY (PARENTAL ANIMALS)
A summary incidence of necropsy findings for adults is given in attached Table 16. Individual data are given in attached Appendix 15.

The male treated with 250 mg/kg bw/day that was found dead on Day 9 did not show any macroscopic abnormalities.
No toxicologically significant macroscopic abnormalities were detected in terminal kill animals of either sex treated with 250 or 100 mg/kg bw/day or males treated with 30 mg/kg bw/day.
One female treated with 30 mg/kg bw/day had a mass in the right thoracic region at necropsy. This was confirmed during microscopic evaluation as an abscess and was considered not to be of toxicological significance.


HISTOPATHOLOGY (PARENTAL ANIMALS)
A complete histopathology report is attached.

The following treatment related microscopic findings were detected:
Liver: minimal centrilobular hypertrophy was evident in animals of either sex treated with 250 mg/kg bw/day and in males treated with 100 mg/kg bw/day. The incidence and severity of glycogen deposit of hepatocytes were also decreased as a consequence of the centrilobular hypertrophy in males treated with 250 mg/kg bw/day.


OTHER FINDINGS (PARENTAL ANIMALS)
WATER CONSUMPTION
Group mean water consumptions are given in attached Table 7. Individual and group mean water consumptions for females following mating and lactation are presented in attached Appendix 5.

There were no adverse effects detected in water consumption for treated animals when compared to control animals.




Dose descriptor:
NOAEL
Effect level:
100
Basis for effect level:
other: other:
Dose descriptor:
NOEL
Effect level:
250
Basis for effect level:
other: other:
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects. Remark: "(under the conditions of the test, number of corpora lutea, implantation rate, postimplantation loss, litter size at first littercheck, body weights of pups or results of external examination gave no indication of embryotoxic or teratogenic effects

Details on embryotoxic / teratogenic effects:
In total all females from the control and 30 mg/kg bw/day dose groups and nine females from the 100 and 250 mg/kg bw/day dose groups gave birth to a live litter and successfully reared young to Day 5 of age. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.

VIABILITY (OFFSPRING)
Group mean corpora lutea and implantation counts, litter size, implantation losses, survival indices and sex ratio are given in Tables 9 to 11. Individual data are given in Appendices 7 to 9.
No toxicologically significant effects were detected.
No significant differences were detected for corpora lutea, implantation counts, litter size or litter viability for treated animals when compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences. Slight intergroup differences were detected in post implantation losses and live birth index at 250 mg/kg bw/day. These were considered to be attributable to isolated litters and in the absence of any significant differences the intergroup difference were considered not to be of toxicological importance.
There were no intergroup differences in sex ratio (percentage male offspring) for litters from treated groups compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.


CLINICAL SIGNS (OFFSPRING)
Group values for surface righting reflex and the incidence of clinical signs are given in attached Tables 12 and 13 attached. Individual data are given in Appendices 10 and 11.
No obvious clinical signs of toxicity were detected for offspring from treated females when compared to controls. The incidental clinical signs detected throughout the control and treated groups, consisting of small size, offspring found dead or missing, bruising, no milk in stomach, cold, weak, swollen abdomen and pale were considered to be low incidence findings observed in offspring in studies of this type, and were unrelated to test item toxicity.The percentage of offspring who successfully showed surface righting reflex on Day 1 was similarly unaffected by maternal exposure.


BODY WEIGHT (OFFSPRING)
Group mean values for total litter weights, offspring body weights and body weight changes are given in attached Table 9. Individual values are given in Appendix 7.
No toxicologically significant effects were detected.
There were no significant differences in litter or offspring weights. Statistical analysis of the data did not reveal any significant intergroup differences.


ORGAN WEIGHTS (OFFSPRING)
Not applicable.

GROSS PATHOLOGY (OFFSPRING)
A summary incidence of necropsy findings for offspring is given in attached Table 15. Individual data is given in attached Appendix 14.
No treatment-related macroscopic abnormalities were detected for interim death or terminal kill offspring. The incidental findings observed were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test item.


HISTOPATHOLOGY (OFFSPRING)
Not examined.

OTHER FINDINGS (OFFSPRING)
Not applicable.

Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
The oral administration of 2,6-Bis(1,1-dimethylethyl)-4-(arylalkylidene)alicycli-2,5-dien-1-one to rats by gavage, at dose levels of 30, 100 and 250 mg/kg bw/day, resulted in treatment related effects detected in animals of either sex treated with 250 mg/kg bw/day.
The microscopic liver changes detected in 100 mg/kg bw/day males and the increased liver weights in females at 100 mg/kg bw/day in isolation were considered to be adaptive in nature and not to represent an adverse effect of treatment. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 100 mg/kg bw/day.
No treatment related effects were detected in the reproductive parameters examined therefore the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 250 mg/kg bw/day.
Executive summary:

Introduction.The study was performed to screen for potential adverse effects of the test item on reproduction including offspring development and provides an initial hazard assessment for effect on reproduction. The study was designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 27 July 1995).

This study was also designed to meet the requirements of Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

The dose levels were chosen based on the results of previous toxicity work (Harlan Laboratories Ltd., Project Number 41101419).

Methods.The test item was administered by gavage to three groups, each of ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week maturation phase, pairing, gestation and early lactation for females), at dose levels of 30, 100 and 250 mg/kg bw/day (adjusting for 12.3% solvent content). A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP).

Clinical signs, body weight change, dietary intake and water consumption were monitored during the study. 

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Survivingadult males were terminated on Day 43, followed by the termination of all females and offspring on Day 5post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of liver and reproductive tissues was performed.

Results.

Adult Responses:

Mortality.One male treated with 250 mg/kg bw/day was found dead on Day 9. There were no further unscheduled deaths.

Clinical Observations.Episodes of increased salivation were evident in animals of either sex from all treatment groups throughout the treatment period. The male treated with 250 mg/kg bw/day that was found dead on Day 9 had orange coloured diarrhoea on Day 4. 

Body Weight.Males treated with 250 mg/kg bw/day showed actual body weight losses during the first week of treatment. A reduction in body weight gain was also evident in these animals during Weeks 3 and 4. Males treated with 100 mg/kg bw/day showed a reduction in body weight gain during the first week of treatment. No such effects were detected in females treated with 250 or 100 mg/kg bw/day or animals of either sex treated with 30 mg/kg bw/day.

Food Consumption and Food Efficiency.Males treated with 250 mg/kg bw/day showed a reduction in food consumption during Week 1 and Week 5. A reduction in food efficiency was also evident in these males during the first week of treatment. 

No toxicologically significant effects were detected in females treated with 250 mg/kg bw/day or animals of either sex treated with 100 or 30 mg/kg bw/day.

Water Consumption. No adverse effect on water consumption was detected.

Reproductive Screening:

Mating. There were no treatment-related effects on mating for treated animals.

Fertility. There were no treatment-related effects on conception rates for treated animals.

Gestation Lengths. There were no differences in gestation lengths. The distribution for treated females was comparable to controls.

Litter Responses:

Offspring Litter Size, Sex Ratio and Viability. Of the litters born, litter size at birth and subsequently on Day 1 and 4post partumwere comparable to controls. There were no intergroup differences in sex ratio.

Offspring Growth and Development. Offspring body weight gain and litter weights at birth and subsequently on Day 1 and 4post partumwere comparable to controls. No effect on surface righting reflex was detected.

Offspring Observations. No clinically observable signs of toxicity were detected for offspring from all treatment groups.

Pathology:

Necropsy. No toxicologically significant macroscopic abnormalities were detected in animals of either sex treated with 250, 100 or 30 mg/kg bw/day.

Organ Weights.Females treated with 250 and 100 mg/kg bw/day showed an increase in liver weight both absolute and relative to body weight.

No such effects were detected in males treated with 250 or 100 mg/kg bw/day or animals of either sex treated with 30 mg/kg bw/day.

No treatment related effects were detected in the reproductive organs weighed in males from any treatment group.

Histopathology.The following treatment related microscopic findings were detected:

Liver:minimal centrilobular hypertrophy was evident in animals of either sex treated with 250 mg/kg bw/day and in males treated with 100 mg/kg bw/day. The incidence and severity of glycogen deposit of hepatocytes were also decreased as a consequence of the centrilobular hypertrophy in males treated with 250 mg/kg bw/day.

Conclusion.The oral administration of 2,6-Bis(1,1-dimethylethyl)-4-(arylalkylidene)alicycli-2,5-dien-1-one to rats by gavage, at dose levels of 30, 100 and 250 mg/kg bw/day,resulted in treatment related effects detected in animals of either sex treated with 250 mg/kg bw/day.

The microscopic liver changes detected in 100 mg/kg bw/day males and the increased liver weights in females at 100 mg/kg bw/day in isolation were considered to be adaptive in nature and not to represent an adverse effect of treatment. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 100 mg/kg bw/day.

No treatment related effects were detected in the reproductive parameters examined therefore the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 250 mg/kg bw/day.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

The oral administration of test substance to rats by gavage, at dose levels of 30, 100 and 250 mg/kg bw/day, resulted in treatment related effects detected in animals of either sex treated with 250 mg/kg bw/day.

 

The microscopic liver changes detected in 100 mg/kg bw/day males and the increased liver weights in females at 100 mg/kg bw/day in isolation were considered to be adaptive in nature and not to represent an adverse effect of treatment. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 100 mg/kg bw/day.

 

No treatment related effects were detected in the reproductive parameters examined therefore the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 250 mg/kg bw/day.


Justification for selection of Effect on developmental toxicity: via oral route:
The NOAEL is derived from systemic toxicity observed in the parent during the OECD 421 study. The NOEL for developmental toxicity was 250 mg/kg bw/day.

Justification for classification or non-classification

The OECD 421Reproduction/Developmental Toxicity Screening Test showed no treatment related effects in the reproductive parameters examined at any dose level. Therefore, there is no evidence that the substance has an adverse effect on sexual function and fertility, or on development. The substance is therefore not classified for reproductive toxicity.