Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 2013 - July 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
Lot number: E11C020
Stability/expiry date: 28 February 2015.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
2.4.1 Animal supply, acclimatisation and allocation
Animals
Strain/Species Crl:CD(SD) rat.
Supplier Charles River (UK) Ltd
Number of animals 128 males and 128 females.
Animals were identified littermates of four of one sex per litter. 32 litters of four males and 32 litters of four females were received and males were not related to females.
Duration of acclimatisation for the F0 animals 12 days before commencement of treatment.
Age of the F0 animals at start of treatment 33 to 39 days old.
Weight range of the F0 animals at the start of treatment Males: 131 g to 194 g
Females: 114 g to 156 g
Health screen
A routine health check was performed after animals were allocated to study. Four spare males (numbers 225-228) and four spare females (numbers 229-232) were selected and were killed, bled and subject to routine macroscopic examination. Serum samples were retained frozen pending possible future serology investigations. Lungs, liver, kidneys, spleen and heart were preserved in fixative.
Results of the health screen necropsy were reviewed; no evidence of specific infectious disease was seen macroscopically.
Allocation (F0 generation)
Allocation During the acclimatisation period, all litters were assessed and animals weighed individually. Animals showing signs of ill health were excluded. Animals were allocated to the F0 generation to ensure not more than one offspring of each sex from each litter was present in each group. Animals at the extreme of the weight range or litters showing large variation in individual weights were not selected.
Identification of animals Each animal was assigned a unique number, and identified using a tail tattoo.
Selection and identification of offspring to form F1 generation
Allocation The offspring with the lowest within-litter identification per sex from each selected litter were selected to form the F1 generation, after exclusion of grossly atypical animals. Where possible, one male and one female were selected from each selected litter.
Selected animals were tattooed on Day 20 of age and separated from littermates on Day 21 of age. Formal commencement of the F1 generation was on a nominal Day 28 of age (where possible 28±2 days of age for selected F1 animals).
Up to two male and two female F1 offspring per group were retained as spares, to provide potential replacement in the event of any mortality. These spares had body weights and clinical signs recorded weekly and were terminated after commencement of the F1 generation.
Identification of selected F1 animals Animals selected for the formal F1 generation animals were assigned a unique number and identified using a tail tattoo
Identification of cages Each cage label was colour-coded according to group. Labels identified the study number, group, cage number and the identity of the occupants
2.4.2 Animal housing, diet and water supply
Environmental control
Rodent facility Full barrier to minimise entry of external biological and chemical agents and to minimise the transference of such agents between rooms.
Air supply Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity Monitored and maintained within the range of 19-23ºC and 40-70%.
There were no deviations from these ranges.
Lighting Artificial lighting, 12 hours light : 12 hours dark.
Electricity supply Public supply with automatic stand-by generators.
Animal accommodation and bedding
Cages Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used throughout the study except during pairing.
Grid bottomed cages (polypropylene) were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
Cage distribution The cages constituting each group were dispersed in batteries so that possible environmental influences arising from their spatial distribution were equilibrated, as far as was practicable.
Bedding Solid bottom cages contained wood based bedding, which was changed at appropriate intervals each week.
Number of animals per cage Pre-pairing (acclimatisation and after selection) up to four animals
Pairing one male and one female animal
Males to termination up to four animals
Females after mating (from Day 0 after mating) one animal
Females during littering (from Day 20 after mating) one animal + litter
Females to termination (after weaning) up to four animals
Environmental enrichment
Aspen chew block Provided to each cage throughout the study (except during pairing and lactation) and replaced when necessary.
Polycarbonate shelter Provided to each cage throughout the study (except during pairing and lactation) and replaced at the same time as the cages.
Diet supply
Diet SDS VRF1 Certified powdered diet.
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Availability Non-restricted.
Water supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability Non-restricted.
Supplier certificates of analysis
Certificates of analysis for the diet are scrutinised and approved before any batch of diet was released for use. Certificates of analysis were routinely provided by the water supplier.
Certificates of analysis were also received from the suppliers of the softwood based bark-free fibre bedding, and Aspen chew blocks.
No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
Test substance administered through the diet, available ad libitum continuously throughout the study
Details on mating procedure:
2.5.5 Mating procedure
F0 pairing commenced After 10 weeks of treatment.
F1 pairing commenced 10 weeks after selection (formal commencement of F1).
Male/female ratio 1:1 from within the same treatment groups (sibling pairing was not permitted).
Duration of pairing Up to two weeks.
Daily checks for evidence of mating Presence of ejected copulation plugs.
Vaginal smear - examined for the presence of spermatozoa and the stage of the oestrous cycle.
Day 0 of gestation When positive evidence of mating was detected.
Male/female separation Day when mating evidence was detected.
Pre-coital interval Calculated for each female as the time between first pairing and evidence of mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
2.2.4 Formulation analysis
Stability and homogeneity The homogeneity and stability of formulations during storage were confirmed as part of another study (Huntingdon Life Sciences Study Number: HJL0174). On that study, homogeneity and stability were confirmed following storage at ambient temperature (nominally 21 °C) for 22 days. Stability was also confirmed following storage at nominally -20 °C, for 43 days.
Achieved concentration At specified intervals during treatment, the test formulations were analysed for achieved concentration of the test substance. On five occasions, approximately 10 weeks apart, four samples (nominally 200 g) were taken from all groups; two assays from each sample. Stored samples were retained as contingency for analysis should any result require confirmation.
Analysis The method of analysis and results are presented in Annex 2.
2.3 Administration
Route Oral, via the diet.
Treated at Constant concentrations in ppm.
Controls (Group 1) Untreated diet of the same batch.
Frequency Continuously.
Diet A record of the usage of the diets was maintained on all occasions when food consumption was measured. This was performed using the initial weight of the diet container and an on-line data check on completion of the feeding procedure to ensure that all cages were fed the correct amount of diet.
Duration of treatment / exposure:
F0 males: 10 weeks until mating commenced, sacrifice at weaning
F0 females: 10 weeks until mating commenced, sacrifice at 28d post partum
F1 generation: Lifetime (at least 10 weeks post-weaning before mating, sacrificed as above)
F2 generation: Lifetime until PND 21
Frequency of treatment:
continuous
Details on study schedule:
Study initiation:
(Protocol signed by Study Director) 14 August 2013

Experimental start date:
(Animal arrival) 28 August 2013

Treatment of F0 animals commenced: 9 September 2013

F1 generation commenced: 9 January 2014

F1 generation necropsy: 13 to 23 May 2014

Experimental completion date:
(Last day of data recording) 02 July 2014
Doses / concentrationsopen allclose all
Dose / conc.:
1 200 ppm
Dose / conc.:
3 500 ppm
Dose / conc.:
12 000 ppm
No. of animals per sex per dose:
F0: 28 males/females per dose
F1: 24 males/females per dose
Control animals:
yes, plain diet

Examinations

Parental animals: Observations and examinations:
2.5.1 Clinical observations
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 occupants. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatisation period, observations of the animals and their cages were recorded at least once per day.
Clinical signs
A detailed weekly physical examination was performed once each week for F0 animals and for F1 animals selected for the formal F1 generation to monitor general health. Females also had a detailed examination on Days 0, 5, 12, 18 and 20 after mating and on Days 1, 7, 14, 21 and 28 of lactation.
Mortality
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.
A complete necropsy was performed in all cases as described in section 2.6.
2.5.2 Body weight
Animals were weighed as follows:
F0 Males Day that treatment commenced (Week 0).
Each week.
At necropsy.
F0 Females Day that treatment commenced (Week 0).
Each week until mating was detected.
Days 0, 7, 14 and 20 after mating.
Days 1, 4, 7, 14, 21, 25 and 28 of lactation.
F1 animals At same frequency as F0 animals following selection (nominally four weeks of age).
More frequent weighings were instituted, when appropriate, for animals displaying ill-health, so that the progress of the observed condition could be monitored. These data are not reported here.
2.5.3 Food consumption
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
F0 males and females: Weekly until paired for mating. From these records the mean weekly consumption per animal (g/rat/week) was calculated for each cage.
F0 females: Days 0-6, 7-13 and 14-19 after mating and Days 1-3, 4-6, 7-13 and 14-20 of lactation. From these records the mean daily consumption (g/rat/day) was calculated for each animal.
F1 animals Recorded at the same frequency as F0 animals following selection, at approximately four weeks of age
Oestrous cyclicity (parental animals):
2.5.4 Oestrous cycles
Dry smears Smears were taken daily for 22 days before pairing, using cotton swabs moistened with saline. The 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.
For four days before scheduled termination (nominally Days 25 to 28 post-partum) daily vaginal smears were taken and used to determine the stage of the oestrous cycle at termination.
For females whose litters had previously died, smears were taken on a theoretical Days 25 28. Females that failed to litter or mate were retained and smeared for four days starting on the day on which the first batch of ‘true’ Day 25 females started smearing; they were then killed with that first batch of females.
Sperm parameters (parental animals):
2.6.1 Sperm analysis
Immediately after scheduled sacrifice of each male, the left vas deferens, epididymis and testis was removed, except for two males (1M 6 and 3M 78) for which tissues were taken from the right side. The epididymis and testis were weighed.
The following tests were performed:
Sperm motility - all groups A sample of sperm was expressed from the vas deferens into pre-warmed (37oC) medium M199, which contained 0.5% w/v bovine serum albumin (BSA Fraction V). A sample for assessment was taken into a 100 µm depth cannula by capillary action and at least 200 sperm per animal analysed using the Hamilton Thorne IVOS Computer Assisted Sperm Analyser (CASA).
Sperm morphology - Group 1 and 4 A 200 L aliquot of the sperm/medium mixture (described above) was diluted with 800 L of 10% neutral buffered formalin. After staining with nigrosine and eosin an air-dried smear was prepared. Slides were examined by light microscopy for the assessment of sperm morphology. Where possible, at least 200 sperm were assessed for each male.
Group 2 and 3 Slides retained for possible future assessment.
Sperm count - Group 1 and 4 The cauda epididymis of each male was weighed; then homogenised for at least one minute in 10 mL of a mixture of 0.9% saline, 0.01% merthiolate and 0.05% Triton X-100 (SMT). An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for sperm count using CASA.
Group 2 and 3 Retained frozen for possible future assessment.
Homogenisation -resistant spermatid count - Group 1 and 4 The testis of each male was homogenised for at least two minutes in 25 mL of SMT. An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for homogenisation-resistant spermatid count using CASA.
Group 2 and 3 Retained frozen for possible future assessment.
Litter observations:
2.5.7 Records made during littering phase (F0 and F1 generation)
The records maintained were as follows:
Clinical signs All litters were examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter.
Offspring identification On Day 1 of age each offspring was numbered individually within each litter using a toe tattoo.
Litter size Daily records were maintained of mortality and consequent changes in litter size from Days 1-21 of age.
On Day 4 of age, litters containing more than ten offspring were reduced to ten by random culling, leaving, whenever possible, five male and five female offspring in each litter.
Sex ratio of each litter Recorded on Days 1, 4 (before and after culling) and on Day 21 of age.
Individual offspring body weights Recorded on Days 1, 4 (before and after culling), 7, 14, 21 and 25 of age.
Weaning of offspring The dam was removed from the litter cage and offspring were weaned on Day 21 of age.
Selection of offspring (F1 generation) The selection of offspring to form the F1 generation was made on Day 18 of age.
Postmortem examinations (parental animals):
The organs weighed, tissue samples fixed and sections examined microscopically are detailed as follows:
Necropsy Histology Pathology
Tissue and regions examined Weigh Fix Light microscopy

ADULT ANIMALS
Abnormalities * * *
Adrenals * * * *
Brain (cerebellum, cerebrum, midbrain) * * # #
Epididymides (caput, corpus and cauda) L+R † † †
Kidneys * * # #
Liver (section from all main lobes) * * # #
Mammary area – caudal b) # #
Ovaries d) L+R L+R L+R L+R
Pituitary * * * *
Prostate * * * *
Seminal vesicles (with coagulation gland) * * * *
Spleen * * # #
Testes L+R † † †
Thyroid with parathyroids a) * # #
Uterus with cervix and oviducts (separate section of each) * * * *
Vagina e) * * *


L+R Bilateral organs weighed individually.
* Organs weighed, samples fixed or sections examined microscopically.
# Examined if effects suspected during the study.
† Only one examined - left testis and epididymis reserved for seminology.
$ Consideration was given to the offspring abnormalities recorded; only those deemed appropriate were examined.
a) Weighed after partial fixation.
b) Females with total litter loss only
c) Procedures for only one male and one female offspring per litter (but abnormalities fixed from all offspring in litter).
d) Fixed identified as L + R. Five sections cut at ca 100 micron intervals from the inner third of each ovary.
e) Section ca 5 mm from vulva.

The retained tissues were checked before disposal of the carcass.

2.6.2 Organ weights
Requisite organs were weighed for all F0 and F1 adult animals killed at scheduled intervals.
For F1 unselected and F2 offspring, one male and one female were selected at random from each litter for organ weights.

2.6.4 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 killed or dying prematurely.
All F0/F1 terminal adult animals of Groups 1 and 4 killed at scheduled termination.
Processing - reproductive organs only The reproductive organs (i.e. the epididymis, prostate, seminal vesicles and testis, or the cervix, ovaries, oviducts, uterus, and vagina) were examined from animals in Groups 2 and 3 that showed reduced fertility. This included males that failed to mate, failed to sire a pregnancy or with abnormal seminology, and females that failed to mate, were not pregnant, failed to litter, or had total litter death.
Abnormalities only All F0/F1 adult animals of Groups 2 and 3 killed at scheduled termination.

All F1 unselected offspring and all F2 offspring.
Routine staining Sections were stained with haematoxylin and eosin.
Postmortem examinations (offspring):
OFFSPRING c)
Abnormalities * $ $
Brain * * # #
Epididymides * # #
Ovaries * # #
Prostate * # #
Seminal vesicles * # #
Spleen * * # #
Testes * # #
Thymus * * # #
Uterus with cervix and oviducts * # #
Vagina * # #
L+R Bilateral organs weighed individually.
* Organs weighed, samples fixed or sections examined microscopically.
# Examined if effects suspected during the study.
† Only one examined - left testis and epididymis reserved for seminology.
$ Consideration was given to the offspring abnormalities recorded; only those deemed appropriate were examined.
a) Weighed after partial fixation.
b) Females with total litter loss only
c) Procedures for only one male and one female offspring per litter (but abnormalities fixed from all offspring in litter).
d) Fixed identified as L + R. Five sections cut at ca 100 micron intervals from the inner third of each ovary.
e) Section ca 5 mm from vulva.
Statistics:
Refer to "any other information on materials and methods" section below
Reproductive indices:
Mating performance and fertility
Individual data was tabulated. Group values were calculated for males and females separately for the following:
Percentage mating = Number animals mating x 100
Animals paired

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

Fertility index (%) = Number animals achieving pregnancy x 100
Animals pairing
Gestation length
Gestation length was calculated as the number of gestation days up to and including the day on which offspring were first observed, with Day 1 = day of mating for calculation purposes. Where parturition had started overnight, this value was adjusted by subtracting half of one day. Gestation index was calculated for each group as:
Gestation index (%) = Number of live litters born x 100
Number pregnant

Offspring viability indices:
Survival indices
The following were calculated for each litter:
Post-implantation survival index (%) = Total number of offspring born x 100
Total number of uterine implantation sites

Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.
Live birth index (%) = Number of live offspring on Day 1 after littering x 100
Total number of offspring born

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

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

Group mean values were calculated from individual litter values.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One P0 female receiving 12000 ppm appeared to complete parturition but was found dead on Day 2 of lactation. This female had 3 dead fetuses with placenta and 7 placenta still attached in the left uterine horn. Endometritis was seen at histopathological examination, and this was considered to be the cause of death of this animal. Since this was a single incidence among F0 females which was not replicated among F1 females receiving 12000 ppm this isolated finding was considered to be unrelated to treatment.

One P0 male receiving 12000 ppm (4M 110) was found dead on Day 114 of treatment. Since there were no histopathological findings or other deaths among males at 12000 ppm in either generation, this single death was considered to be unrelated to treatment.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Among adult P0 and P1 generations exposed to test article, bodyweight gain was low for periods throughout the pre-pairing period and food intake was low during Week 6-8 for P0 males when compared to Controls. These effects on bodyweight and food consumption were not consistent and are therefore not considered adverse as they had no overall effect on growth, clinical condition, survival or reproductive performance.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption was unaffected during the pre-pairing phase of treatment when compared with Controls.
Food intake was slightly but statistically significantly lower during Weeks 6-8 for males and statistically significantly higher during Weeks 8 and 10 for females receiving 12000 ppm, when compared with Controls.
Group mean food consumption during gestation and lactation was considered unaffected by treatment with test article, when compared with Controls.
Food intake was slightly but statistically significantly higher during Days 14-19 of gestation for females receiving 12000 ppm, when compared with Controls.
Food efficiency:
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
One P0 female receiving 12000 ppm was observed with total litter resorption. This was not considered to be treatment related as it was not replicated in the P1 generation, where greater exposure magnitude and duration to the test article compared to that of the P0 generation. If this finding was treatment related, total litter resorption should also have been apparent in F2 litters.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
12 000 ppm
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Among adult P0 and P1 generations exposed to test article, bodyweight gain was low for periods throughout the pre-pairing period and food intake was low during Week 6-8 for P0 males when compared to Controls. These effects on bodyweight and food consumption were not consistent and are therefore not considered adverse as they had no overall effect on growth, clinical condition, survival or reproductive performance.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption was unaffected during the pre-pairing phase of treatment when compared with Controls.
Food intake was slightly but statistically significantly lower during Week 6 before pairing for females receiving 12000 ppm, when compared with Controls.
Group mean food consumption during gestation and lactation was considered unaffected by treatment with test article, when compared with Controls.
Food efficiency:
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no differences in P0/P1 adult organ weights which were considered to be toxicologically significant. Absolute adrenal weights among P0 and P1 males receiving 12000 ppm were statistically significantly lower than in Controls but there were no such differences in body weight relative weights and no treatment related macroscopic or microscopic changes were detected in this organ. Body weight relative ovary weights for P0 females receiving 3500 or 12000 ppm were statistically significantly lower than in Controls but this difference was not apparent among P1 females and no treatment related macroscopic or microscopic changes were detected in this organ. Among P0 males receiving 12000 ppm, absolute and body weight relative liver weight was statistically significantly lower than in Controls but this difference was not apparent among P1 males and no effect of treatment was inferred. Among all groups of P1 females receiving test article, body weight relative brain weight was statistically significantly higher than in Controls but there was no relationship to dietary concentration. Since differences from Control were small (< 7%) and treatment did not affect the survival, clinical condition or reproductive performance of these females, this finding was considered not to represent an adverse effect of treatment.
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No macroscopic or histopathological treatment-related changes were evident in either phase (P0 or P1) in the animals examined. In most animals with suspected fertility issues there was no histopathological correlation. In the few animals with correlating microscopic findings (1M 315, 4M 377, 1F 416 and 2F 431) the changes were consistent with background findings seen in other studies.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
Two neoplastic findings were seen in P1 animals killed at the end of the treatment period. A mammary adenocarcinoma was found in a Control male (1M 309) and a mammary fibroadenoma in a 3500 ppm-exposed female (2F 428). These tumours were considered to be incidental and unrelated to treatment with the test article.

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
One P0 female receiving 12000 ppm was observed with total litter resorption. This was not considered to be treatment related as it was not replicated in the P1 generation, where greater exposure magnitude and duration to the test article compared to that of the P0 generation. If this finding was treatment related, total litter resorption should also have been apparent in F2 litters.

Effect levels (P1)

Dose descriptor:
NOAEL
Effect level:
>= 12 000 ppm
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean bodyweight gains of males and females in all groups treated with test substance were marginally lower than in Controls but there was no dose response and this was considered to relate to the slightly higher litter size (and thus greater within litter competition between pups) in these groups, since there was no effect of treatment on the total performance (live litter size on Day 1 x mean weight gain Days 1-21) of the male and female pups in the litters
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Ophthalmological findings:
no effects observed
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:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 12 000 ppm
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Results: F2 generation

General toxicity (F2)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean bodyweight gains of males and females in all groups treated with test substance were marginally lower than in Controls but there was no dose response and this was considered to relate to the slightly higher litter size (and thus greater within litter competition between pups) in these groups, since there was no effect of treatment on the total performance (live litter size on Day 1 x mean weight gain Days 1-21) of the male and female pups in the litters.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The absolute brain weights of male offspring in the treated groups with the exception of 1200 ppm group which had the lowest terminal bodyweight, were, as expected, and similar to Controls despite the lower terminal bodyweights in these groups; as a consequence, mean bodyweight relative brain weights were marginally but significantly higher than in Controls but this is an expected observation and of no toxicological importance.

Absolute thymus weights were slightly but statistically significantly lower than Controls for the 1200, 3500 and 12000 ppm groups in both sexes, with statistical significance being attained for mean bodyweight relative thymus weights for male offspring in the 12000 ppm group. The aetiology of this is unknown as no pathological assessment has been conducted on the offspring thymus, but this finding is of an uncertain relationship to treatment as there was no dose related response and no such difference was apparent in females, and the Control value is slightly higher than the background control data.
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Other effects:
not specified

Developmental neurotoxicity (F2)

Behaviour (functional findings):
no effects observed

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Effect levels (F2)

Dose descriptor:
NOAEL
Generation:
F2
Effect level:
>= 12 000 ppm
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
The NOAEL for reproductive effects and systemic toxicity is >=12000 ppm based on absence of treatment related adverse effects at the highest tested dose.
Executive summary:

No signs of systemic toxicity or effects on reproductive performance occurred in rats exposed by diets containing up to 12000 ppm of the test article.

Sprague-Dawley rats were exposed to the test article at dietary concentrations of 0, 1200, 3500, or 12000 ppm (with mean achieved dosages of 89-102, 257-300 or 868-1030 mg/kg bw/d for males [P0 and F1/P1] and 87-214, 262-640 and 934-2129 mg/kg bw/d for females [P0 and F1/P1 generations across all stages of pregnancy]) in an GLP-compliant OECD 416 test guideline Two Generation Reproductive Performance study.

There were no effects of treatment on oestrous cycles, pre-coital interval, mating performance, fertility, gestation lengths and gestation index, sperm motility, concentrations or morphology, for either generation at dietary concentrations up to 12000 ppm.

There was no effect of treatment on the ability of the P0 or F1/P1 females to successfully litter and rear their offspring to weaning. Litter size, survival of the offspring, general condition, offspring bodyweight and bodyweight gain and the offspring sex ratio were not affected by treatment of either generation.

There were no toxicologically significant differences in P0 or P1 adult organ weights. No macroscopic or histopathological treatment-related changes were evident in either phase (P0 or F1/P1) in the animals examined.

F2 offspring absolute thymus weights were slightly but statistically significantly lower than Controls for the 1200, 3500 and 12000 ppm groups in both sexes, with statistical significance being attained for mean bodyweight relative thymus weights for male offspring in the 12000 ppm group. The aetiology of this is unknown as no pathological assessment has been conducted on the offspring thymus, but this finding is not considered adverse as there was no dose related response, and no effect on the survival, clinical condition or growth of the animals, and the control value was higher than the background control data.