Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-07-17 to 2018-09-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Wistar Han™:RccHan™:WIST strain rats
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: [yes/no]
- Age at study initiation:
males: approximately eleven weeks old and females: approximately twelve weeks old.
- Weight at study initiation:
males: 283 to 367g and females: 190 to 232g
- Fasting period before study:
- Housing:
in groups of three in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding
- Diet (e.g. ad libitum):
free access
- Water (e.g. ad libitum):
free access
- Acclimation period:
twenty days

DETAILS OF FOOD AND WATER QUALITY:
A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.) was used. Certificates of analysis of the batches of diet used are given in the original report. Mains drinking water was supplied from polycarbonate bottles attached to the cage.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
22 ± 3 °C
- Humidity (%):
50 ± 20%
- Air changes (per hr):
at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light):
12 hrs dark / 12 hrs light

IN-LIFE DATES: From: To: The in-life phase of the study was conducted between 17 July 2018 (first day of treatment) and 18 September 2018 (final day of necropsy).

Humidity:
The Study Plan target value for relative humidity (RH) was 50 ± 20%. This target range was exceeded on several occasions during the study with a maximum relative humidity of 74% being reached. Whilst these deviations from the target range were regrettable, the condition of the animals was closely monitored and there was no indication that the incidence of high humidity had any impact on the health of the animals. It was therefore considered that the purpose and integrity of the study had been unaffected.



Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Dried Propylene Glycol
Details on exposure:
The test item was administered daily by gavage using a rubber dosing catheter attached to a graduated disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Dried Propylene Glycol.
Details on mating procedure:
- M/F ratio per cage: 1 male: 1 female basis
- Length of cohabitation: for a period of up to fourteen 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 estrus 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 (how): 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.





Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of test item formulations were taken in two occasions and analyzed for concentration of the substance. The results indicate that the prepared formulations were within 95-100% of the nominal concentration indicating acceptable accuracy of the formulation procedure.
Duration of treatment / exposure:
Four weeks for males and at least seven weeks for females (including a two week pre-pairing phase, pairing, gestation and lactation phases)
Frequency of treatment:
once/day
Details on study schedule:
Please see under section "Any other information on materials and methods incl. tables”
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
750 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Not specified

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
immediately before dosing, soon after dosing, and one hour after dosing during the working week (except for females during parturition where applicable).

Morbidity/Mortality inspections were scheduled in the Study Plan to take place, twice daily, early and late during the working period. On three occasions (twice during the acclimatization period and on the penultimate day of the in-life phase of the study), the morning check of the animals was performed later than normally considered acceptable. Whilst this is regrettable, there was no indication that this resulted in any delay in detecting effects on the animal’s health during the study and as such, there was no impact on the scientific integrity of the study.

Post-dosing observations were not recorded on Day 26 of the study in error. Based on the post-dosing observation observed both prior to and after this occurrence, it is considered that no significant clinical signs would have been missed, due to this oversight. This deviation is, therefore, considered to have had no impact on the scientific integrity of the study.

BODY WEIGHT: Yes
- Time schedule for examinations:
On Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1, 4, 7 and 13 post partum. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
During the pre-pairing period, weekly food consumption was recorded for each cage of adults. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded for the periods covering post partum Days 1-4, 4-7 and 7-13.

FOOD EFFICIENCY:
%Food Efficiency = (Group mean bodyweight gain (g/rat/period) / Group mean food consumption (g/rat/day) x number of days) x 100
Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males and females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations:
Water intake was measured gravimetrically on a daily basis from Day 2 of the study throughout the pre-pairing period.

OPHTHALMOSCOPIC EXAMINATION: Not specified

HAEMATOLOGY: Yes
Hematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 28 for males and Day 13 post partum for females). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling.
- Parameters checked in table [No.4] were examined.

CLINICAL CHEMISTRY: Yes
Hematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 28 for males and Day 13 post partum for females). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling.
- Parameters checked in table [No.5] were examined.

URINALYSIS: No


BEHAVIORAL ASSESSMENT: Yes
- Time schedule for examinations:
Prior to the start of treatment and at approximately weekly intervals thereafter
- Dose groups that were examined:
all
- Parameters checked in table [No.7] were examined.

IMMUNOLOGY: No

PREGNANCY AND PARTURITION.
Each pregnant female was observed at least three times a day (early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition. Observations were carried out at approximately 0830 and as late as possible at weekends and public holidays. The following was recorded for each female:
- Date of pairing
- Date of mating
- Date and time of observed start of parturition
- Date and time of observed completion of parturition


OTHER:
Functional observations:
Prior to the start of treatment and at approximately weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. These observations were performed on mated females on Days 4, 11 and 18 post coitum and for littering females on Days 4 and 12 post partum. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.
No functional observations were performed for Females 38 and 43 on Day 18 of gestation in error. Although this loss of data is regrettable, there is considered to be sufficient data from the remaining animals to draw meaningful conclusions from. The purpose and scientific integrity of the study is therefore unaffected.
Oestrous cyclicity (parental animals):
Vaginal smears were performed for all females from the day after arrival (enabling the exclusion of females not showing appropriate estrous cycling from dosing). Vaginal smears were taken daily for females throughout the two week pre-pairing treatment period and in the morning of the day of necropsy. The stage of the estrous cycle was recorded for each day.

Sperm parameters (parental animals):
Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell-or stage-specificity of testicular findings was noted.
Litter observations:
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.
For each litter the following was recorded:
- Number of offspring born
- Number of offspring alive recorded daily and reported on Days 1, 4, 7 and 13 post partum
- Sex of offspring on Days 1, 4 and 13 post partum
- Clinical condition of offspring from birth to Day 13 post partum
- Individual offspring weights on Days 1, 4, 7 and 13 post partum (litter weights were calculated retrospectively from this data)

Physical Development:
All live offspring were assessed for ano-genital distance on Day 1 post partum. Additionally, visible nipple count was performed for all male offspring on Day 13 post partum.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
Adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 29. Adult females were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 12 post partum. All adult animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964).

HISTOPATHOLOGY: Yes (see table 1)
Samples of the following tissues were removed from five selected males and five selected females from each dose group and preserved in buffered 10% formalin, except where stated.

Postmortem examinations (offspring):
Surviving offspring were terminated by carbon dioxide asphyxiation followed by cervical dislocation on Day 13 post partum. Offspring required for blood sampling were terminated by cervical dislocation with death confirmed by decapitation during the sampling procedure with blood samples collected immediately following decapitation.
Offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. Examination of offspring was restricted to a macroscopic external examination except where abnormalities were observed, then an additional internal examination was performed.
Statistics:
Data on statistics is stated in section "Any other information on materials and methods incl. tables"
Reproductive indices:
Mating Performance and Fertility
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 x 100
Gestation and Parturition Data
The following parameters were calculated from individual data during the gestation and parturition period of the parental generation:

- Gestation Length

Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.

- Parturition Index

The following was calculated for each group:

Parturition Index (%) = Number of females delivering live offspring / Number of pregnant females x 100
Offspring viability indices:
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 13 of age).
- Implantation Losses (%)
Group mean percentile post-implantation loss was calculated for each female/litter as follows:

Post–implantation loss (%) = Number of implantation sites / Total number of offspring born/Number of implantation sites x 100

- Live Birth and Viability Indices
The following indices were calculated for each litter as follows:

Live Birth Index (%) = Number of offspring alive on Day 1 / Number of offspring born x 100

Viability Index 1 (%) = Number of offspring alive on Day 4 / Number of offspring alive on Day 1 x 100

Viability Index 2 (%) = Number of offspring alive on Day 13 / Number of offspring alive on Day 4 x 100

Viability index 2 takes into consideration the offspring used for blood sampling on Day 4 post partum.

- Sex Ratio (% males)
Sex ratio was calculated for each litter value on Days 1, 4, 7 and 13 post partum, using the following formula:

Number of ofmale offspring / Total number of offspring x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
Instances of increased post-dosing salivation were observed for the majority of animals (10/12 males and 11/12 females) receiving 750 mg/kg bw/day.

Instances of increased post-dosing salivation were observed for the majority of animals (10/12 males and 11/12 females) receiving 750 mg/kg bw/day. The incidence of this clinical sign at this dosage appeared to be slightly higher for females than for males , but this may have reflected the comparatively longer treatment period for females compared to their male counterparts. Increased post-dosing salivation was also observed for the majority of animals (7/12 males and 8/12 females) receiving 300 mg/kg bw/day, although the incidence of this sign, which was similar in both sexes, appeared lower than that observed at 750 mg/kg bw/day. At 100 mg/kg bw/day, increased post-dosing salivation was restricted to just two females, each on only a single occasion towards the end of their treatment period. Increased post-dosing salivation is often observed when animals are dosed via the oral gavage route and are generally considered to reflect slight distaste or irritancy of the test item rather than a systemic effect of treatment.
Occasional instances of noisy respiration were observed for two females at 300 mg/kg bw/day and three males and four females at 750 mg/kg bw/day during the study, with one of the females at 750 mg/kg bw/day also showing noisy respiration during the assessment of behavioral observations. Additionally, one female at 100 mg/kg bw/day and one male at 750 mg/kg bw/day showed noisy respiration during the assessment of behavioral observations. Although the incidence of noisy respiration was higher at the high dosage of 750 mg/kg bw/day, the instances of noisy respiration tended to be quite isolated and, overall, the occurrence of this clinical sign was considered to reflect occasional difficulties in dosing isolated animals and to be of no toxicological significance .
One female treated with 300 mg/kg bw/day showed generalized fur loss from Day 24 of the study, but, in isolation, this finding was considered to be incidental and unrelated to treatment.
Summary incidence of daily clinical obserbations are given in table 6.
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Group mean weekly body weights and standard deviations are given in Table 2 and 3 stated in the key robust study summary "repeated dose toxicity: oral_2019" . Group mean weekly body weight gains and standard deviations are given in Table 4 to 6 stated in the key robust study summary "repeated dose toxicity: oral_2119".
Males
At 750 mg/kg bw/day body weight gain of males tended to be lower than control throughout much of the treatment period, with differences attaining statistical significance during Weeks 2 and 4, and leading to statistically significantly lower overall body weight gain at termination on Day 29 of the study. There was no effect of treatment on body weight gain for males throughout the study at dosages of 100 or 300 mg/kg bw/day.
Females
There was no effect of treatment on body weight and body weight gain for females during the pre-pairing phase of the study at 100, 300 or 750 mg/kg bw/day.
At 750 mg/kg bw/day, mean body weight gains during the last week of gestation was slightly lower than control, leading to lower overall body weight gain, although differences from control failed to attain statistical significance. During the last week of treatment, body weight gain of the pregnant females is increasingly influenced by the growing litter and the lower gain observed at this dosage probably reflect lower litter weight/contribution from the gravid uterus rather than any underlying effect on maternal body weight. Supporting this, mean body weight on Day 1 of lactation was similar to control.
At 750 mg/kg bw/day, mean body weight gain of females tended to be lower than control throughout lactation, with cumulative body weight gain being statistically significantly lower than control on Days 7 and 14 of lactation.
Body weight gains during pregnancy and lactation was unaffected by treatment at 100 or 300 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Group mean food consumptions are given in Table 7 and 8 stated in the key robust study summary "repeated dose toxicity: oral_2019".

Males
There was no effect of treatment on food consumption of males during the pre-pairing phase of the study at 100, 300 or 750 mg/kg bw/day.
Females
There was no effect of treatment on food consumption of females during the pre-pairing phase of the study at 100, 300 or 750 mg/kg bw/day.
At 750 mg/kg bw/day, mean food consumption tended to be lower than control throughout gestation, although statistical significance was only apparent during the first week of pregnancy. Subsequent mean food consumption throughout lactation was statistically significantly lower than control at this dosage.
At 100 and 300 mg/kg bw/day, mean food consumption during pregnancy and lactation was essentially similar to control and unaffected by treatment at either dosage.
Food efficiency:
no effects observed
Description (incidence and severity):
Food efficiency for males and for females during the pre-mating phase is given in Table 9 stated in the key robust study summary "repeated dose toxicity: oral_2019".

Males
At 750 mg/kg bw/day, food conversion efficiency was slightly lower than control during the second week of treatment, leading to slightly lower overall food conversion efficiency for the two week pre-pairing period, compared to control.
There was no effect of treatment on food conversion efficiency of males during the pre-pairing phase of the study at 100 or 300 mg/kg bw/day.
Females
There was no effect of treatment on food conversion efficiency of females during the pre-pairing phase of the study at 100, 300 or 750 mg/kg bw/day.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Water consumption of either sex was considered to be unaffected by treatment at 100, 300 or 750 mg/kg bw/day.

There was no clear effect of treatment on water consumption during the pre-pairing treatment phase for either sex at 100 300 or 750 g/kg bw/day.
For females at 750 mg/kg bw/day water consumption appeared higher than control during the last five days of treatment but there was considered to be insufficient consistency between the individual cages at this dosage to indicate a treatment- related effect.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Intergroup differences for hematology parameters at the end of the treatment period did not indicate any effect of treatment for either sex at 100, 300 or 750 mg/kg bw/day.
For females at 750 mg/kg bw/day, mean reticulocytes counts were statistically significantly higher than control, with 4/5 individual values exceeding the historical control. In the absence of any supporting statistically significant differences from control for other erythrocyte parameters or any supporting histopathological change, this isolated finding was considered to be incidental and unrelated to treatment.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There was no effect of treatment on blood chemistry parameters for either sex at 100, 300 or 750 mg/kg bw/day.

Intergroup differences for blood chemistry parameters at the end of the treatment period did not indicate any adverse effect of treatment for either sex at 100, 300 or 750 mg/kg bw/day.
For males at 300 and 750 mg/kg bw/day, mean bile acid levels were statistically significantly higher than control, with 2/5 individual values at 300 mg/kg bw/day and 3/5 individual values at 750 mg/kg bw/day exceeding the historical control group. In the absence of any supporting histopathological change, this isolated finding was considered to be of no toxicological significance.
For males at 750 mg/kg bw/day, mean alanine aminotransferase activity was statistically significantly higher than control, but only 1/5 individual values for these treated animals were within the historical control range. In the absence of any supporting histopathological change, this isolated finding was considered to be incidental and unrelated to treatment.
For females at 100 and 300 mg/kg bw/day, mean albumin/globulin (A/G) ratio was statistically significantly lower than control, however, there was no corresponding statistically significant differences apparent for mean levels of total protein or albumin at these dosages. All individual values for the A/G ratio of these treated females were within the historical control range and, in the absence of any similar effect at 750 mg/kg bw/day or supporting histopathological change, this finding was considered to be incidental and unrelated to treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
- Behavioral Assessment
Behavioral assessment throughout the study did not indicate any effect of treatment at 100, 300 or 750 mg/kg bw/day.
At 100 mg/kg bw/day, noisy respiration was observed for one female during the second week of lactation. At 750 mg/kg bw/day, noisy respiration was observed for one male during Week 5 and one female during the first week of lactation. These isolated instances of noisy respiration were consistent with similar instances observed during routine assessments of clinical condition and were considered to reflect occasional difficulties in dosing isolated animals. As such, they were considered not to indicate any underlying neurological effect on the behavior of the animals.
- Functional performance
Functional performance tests did not indicate any obvious effect of treatment for either sex at 100, 300 or 750 mg/kg bw/day.
At 100 mg/kg bw/day, overall motor activity for males was statistically significantly higher than control, however in the absence of any similar effect at higher dosages, this finding was considered incidental and unrelated to treatment.
- Sensory reactivity assessments
Sensory reactivity assessments did not indicate any effect of treatment for either sex at 100, 300 or 750 mg/kg bw/day.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Microscopic examination of tissues did not indicate any effect of treatment at 750 mg/kg bw/day.

Adrenal Gland
Hypertrophy of the zona glomerulosa was present at a minimal level in 2/5 females treated at 750 mg/kg bw/day. Due to the low severity and the fact that the animals were lactating it is considered that this change is likely to be due to individual variation. Change in the zona glomerulosa is likely to be an adaptive response. The zona glomerulosa is the site of synthesis of aldosterone a mineralocorticoid which is mainly involved in the control of salt and water balance in the body.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
Microscopic examination of tissues did not indicate any effect of treatment at 750 mg/kg bw/day.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Group mean values and standard deviations for for T4 and T3 in test and control group animals are given in Table 10 and Table 11, respectively stated in the key robust study summary "repeated dose toxicity: oral_2019".
At 750 mg/kg bw/day, mean thyroxine (T4) levels for adult males were lower than control, however, in the absence of any effect on organ weight or supporting histopathological change, this isolated finding was considered to be incidental and of no toxicological significance. No statistically significant differences from control were apparent for triiodothyronine (T3) levels for these adult males or for T4 or T3 levels in Day 13 offspring of either sex at this dosage.
Evaluation of T4 and T3 in adult males and offspring at Day 13 of age did not identify any obvious effect of treatment or indication of endocrine disruption at 100 or 300 mg/kg bw/day.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
A summary of adult performance is presented in Table 1
Estrous cycles during the two week pre-pairing treatment phase and the stage of estrous at terminal necropsy were unaffected by treatment at 100, 300 or 750 mg/kg bw/day.

Other: There were no test item-related microscopic findings in the reproductive tracts following the qualitative examination of the evaluation of the uterus or of follicles and corpora lutea in the ovaries.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic findings in the reproductive tracts following the qualitative examination of the stages of spermatogenesis in the testes (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle).
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Mating:
A summary of adult performance is presented in Table 1. A summary incidence for mating performance is presented in Table 2.
Mating performance, as assessed by the number of paired animals that mated, was unaffected by treatment at dosages of 100, 300 or 750 mg/kg bw/day.
At 750 mg/kg bw/day, although the majority of females mated within the first four days of pairing, which normally indicates the first oestrus opportunity, two females mated on Day 6 and another female mated on Day 14. While, this finding is unusual, it still represented the first oestrus opportunity for these particular females based on the smearing records and, in the absence of any supporting histopathological change in the female reproductive organs, this finding was considered to be unrelated to treatment. For the female that mated very late in the pairing period, it is considered that the animal had probably initially gone into pseudopregnancy (this can occasionally happen when the animals are being smeared) and this was the underlying reason for the extended pre-coital interval.

Fertility:
Group values for fertility, litter data and implantation losses are given in Tables 2, 3 and 7.
There was no effect on fertility, as assessed by the number of females that achieved pregnancy, at 100, 300 or 750 mg/kg bw/day.

Gestation Length
A summary of gestation lengths is presented in Table 2. I
The intergroup distribution of gestation lengths observed during the study did not indicate any obvious effect of treatment at 100, 300 or 750 mg/kg bw/day.
At 300 and 750 mg/kg bw/day, there was tendency towards longer gestation lengths compared with control, with the distribution of gestation lengths attaining statistical significance when compared to control. However, the gestation lengths observed for these treated groups were all unremarkable and within normal expectations. This finding was, therefore, considered to be incidental and unrelated to treatment.

There were several non-productive matings within the study, affecting all groups including control. No changes were present to account for the lack of fertility in any animals.

Effect levels (P0)

open allclose all
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
other: food consumption
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absence of clear adverse effect of treatment for general toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absence of clear adverse effect of treatment for reproduction

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
Clinical signs apparent for the offspring during the study were generally typical of the age observed and the distribution and incidence of these findings did not indicate any obvious effect of maternal treatment on offspring development. At 750 mg/kg bw/day, there was a higher incidence of small offspring observed during Days 5-7 and 8 to 13; compared to control; this finding was consistent with the lower offspring growth, as indicated by lower offspring weight gain, at this dosage, compared to control.
Dermal irritation (if dermal study):
not specified
Mortality / viability:
no mortality observed
Description (incidence and severity):
There were no unscheduled deaths during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 750 mg/kg bw/day, mean body weight gain of the offspring was lower than control from Day 1 to termination on Day 13 of age, with differences attaining statistical significance for males by Day 4 of age and females by Day 7 of age. This lower gain occurred despite the slightly lower litter size at this dosage, compared to control, and resulted in statistically significantly lower mean offspring body weights being apparent by Day 7 of age. Litter weights were statistically significantly lower than control from Day 4, reflecting both the lower body weight gain and the slightly lower litter size compared to control at this dosage.
See table 4.
There was no effect of maternal treatment on mean offspring body weight on Day 1 and subsequent mean offspring body weight gain to Day 13 at 100 or 300 mg/kg bw/day. Litter weights were unaffected by maternal treatment at both dosages.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
See table 8
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic necropsy findings for offspring on the study were typical for the age observed and neither the incidence nor the distribution of these observations indicated any effect of maternal treatment on offspring development at 100, 300 or 750 mg/kg bw/day. At 750 mg/kg bw/day, there was a higher incidence of small offspring observed; this finding was consistent with the lower offspring growth, as indicated by lower offspring weight gain, compared to control, at this dosage.
Histopathological findings:
not specified
Other effects:
no effects observed
Description (incidence and severity):
No statistically significant differences from control were for T4 or T3 levels in Day 13 offspring of either sex at this dosage. See table 10 and 11 stated in the key robust study summary "repeated dose toxicity: oral_2119".

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

open allclose all
Dose descriptor:
NOEL
Generation:
F1
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
body weight and weight gain

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Litter responses:

One control female failed to mate and there were 2, 0, 2 and 1 750 mg/kg bw/day mated females that failed to achieve pregnancy in the control, 100, 300 and 750 mg/kg bw/day dosage groupsrespectively. Additionally one female at 100 mg/kg bw/day shwed total litter losspost partum. The assessment is generally based on the 9, 11, 10 and 11 females successfully rearing young to Day 13 of age at 0 (Control), 100, 300 and 750 mg/kg bw/dayrespectively.

Offspring Litter Size, Sex Ratio and Viability:

Group mean implantation counts, litter size, implantation losses, survival indices and sex ratio are given in Tables 3, 7 and 8.

There was considered to be no effect of maternal treatment on the numbers of implantations, post-implantation loss, litter size and sex ratio at birth/Day 1 and subsequent offspring survival and sex ratio to Day 13 of age at 100, 300 or 750 mg/kg bw/day.

At 750 mg/kg bw/day, the mean number of implantations was slightly lower than control and this subsequently lead to lower litter size from Day 1, however these differences from control failed to attain statistical significance and were considered to reflect normal biological variation.  

Table1     Summary of Reproductive Performance - Group Values

 

Dose Group (mg/kg bw/day)

 

0 (Control)

100

300

750

Males

 

 

 

 

Initial group size

12

12

12

12

Paired

12

12

12

12

Non-mated

1

0

0

0

Induced pregnancy in female partner

9

12

10

11

Surviving to terminal necropsy

12

12

12

12

 

 

 

 

 

Females

 

 

 

 

Initial group size

12

12

12

12

Paired

12

12

12

12

Non-mated

1

0

0

0

Non-pregnant

2

0

2

1

Total Litter Losspost partum

0

1

0

0

Rearing young to Day 13 of age

9

11

10

11

Table 2          Summary Incidence of Mating Performance, Fertility and Gestation Lengths

Group

Number of Males Paired

Number of Females

Pre-Coital Interval (Days)

Mating Index (%)

PregnancyIndex
(%)

Gestation Length (Days)

Females with live Offspring

Parturition Index (%)

Paired

Mated

Pregnant

1

2

3

4

6

14

22½

23

23½

24

1

12

12

11

9

3

2

4

2

0

0

92

82

8

1

0

0

9

100

2

12

12

12

12

4

2

3

3

0

0

100

100

8

2

1

1#

12#

100#

3

12

12

12

10

4

4

2

2

0

0

100

83

2

5

3

0**

10

100

4

12

12

12

11

3

2

4

0

2

1

100

92

0

4

7

0***

11

100

Statistical analysis (distribution of pre-coital interval and gestation length)

# Includes one female with total litter losspost partum

Dose Levels: Group 1 ƒ 0 (Control)   Group 2 ƒ100mg/kg bw/day   Group 3 ƒ300mg/kg bw/day   Group 4 ƒ750mg/kg bw/day            

Table3   Group Mean Litter Size

Group

 

Number of Implantation Sites

Total Number of Offspring Born

Number of Live Offspring

 

 

 

Day 1

Day 4 BC

Day 4 AC

Day 7

Day 13

 

 

 

1

Mean

10.8

10.3

10.3

10.3

8.9

8.9

8.9

 

 

 

S.D.

2.4

2.1

2.1

2.1

1.4

1.4

1.4

 

 

 

N

9

9

9

9

9

9

9

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2

Mean

12.5

12.1

12.1

11.2

9.6

9.6

9.5

 

 

 

S.D.

2.9

3.2

3.2

3.4

2.9

2.9

2.7

 

 

 

N

11

11

11

11

11

11

11

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

3

Mean

11.5

10.7

10.4

10.3

8.9

8.9

8.9

 

 

 

S.D.

3.3

3.2

3.0

2.8

2.1

2.1

2.1

 

 

 

N

10

10

10

10

10

10

10

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

4

Mean

9.5

8.9

8.9

8.5

7.5

7.4

7.4

 

 

 

S.D.

3.5

3.2

3.2

3.1

2.3

2.2

2.2

 

 

 

N

11

11

11

11

11

11

11

 

 

 

Dose Levels: Group 1 ƒ 0 (Control)   Group 2 ƒ 100 mg/kg bw/day   Group 3 ƒ 300 mg/kg bw/day   Group 4 ƒ 750 mg/kg bw/day

Table 4          Group Mean Litter Values for Offspring Weights and Litter Weights

Group

 

                          Offspring Weight (g)

Litter weight (g)

Day 1

Day 4 BC

Day 4 AC

Day 7

Day 13

 

 

 

 

 

Males

Females

Males

Females

Males

Females

Males

Females

Males

Females

Day 1

Day 4 BC

Day 4 AC

Day 7

Day 13

1

Mean

5.87

5.46

8.25

7.64

8.24

7.70

12.55

11.79

24.31

23.05

58.56

82.56

71.43

109.09

212.12

S.D.

0.48

0.33

0.84

0.65

0.84

0.68

1.08

0.80

1.74

1.40

7.80

13.53

9.54

14.11

28.60

N

9

8

9

8

9

8

9

8

9

8

9

9

9

9

9

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2

Mean

5.98

5.63

8.76

8.34

8.77

8.36

13.46

13.05

25.49

24.67

68.21

92.17

79.88

123.74

232.14

S.D.

0.88

0.80

1.39

1.34

1.38

1.32

1.85

1.84

2.68

2.72

15.21

21.03

16.82

25.65

50.87

N

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

3

Mean

6.29

5.91

8.97

8.51

8.96

8.55

13.51

13.06

25.52

24.59

62.26

88.16

76.69

116.22

218.16

S.D.

0.53

0.50

0.89

0.93

0.90

0.96

1.03

1.45

1.92

2.71

15.60

21.87

16.33

24.18

40.86

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

4

Mean

5.64

5.34

7.26

6.92

7.26

6.88

10.20*

9.91*

19.56**

19.07**

48.29

58.37*

51.49*

72.15**

139.27**

S.D.

0.51

0.40

1.20

1.03

1.21

1.09

1.92

1.58

3.41

2.44

16.05

20.91

15.18

20.96

40.20

N

11

11

11

11

11

11

11

11

11

11

11

11

11

11

11

Dose Levels: Group 1 ƒ 0 (Control)   Group 2 ƒ 100 mg/kg bw/day   Group 3 ƒ 300 mg/kg bw/day   Group 4 ƒ 750 mg/kg bw/day  

Table 5          Summary Incidence of Clinical Signs for Offspring          

 

Dose Level (mg/kg bw/day)

 

0 (Control)

100

300

750

Number of litters ()

 

 

 

 

 

 

 

 

 

Birth to Day 4

 

 

 

 

Small

1F (1)

 

1M 1F (2)

1M 3F (2)

Found dead

 

1F (1)

2M (2)

1M 3F (2)

Missing

 

3M 7F (2)

2U (1)

1M (1)

Cold

 

 

1F (1)

4M 5F (3)

Bruise on snout

 

 

 

1M (1)

Weak

 

 

 

2F (2)

 

 

 

 

 

Day 5 to Day 7

 

 

 

 

Small

3M 2F (2)

2M 2F (2)

2F (2)

10M 11F (6)

Pale

1F (1)

 

 

 

Missing

 

 

 

1F (1)

 

 

 

 

 

Day 8 to Day 13

 

 

 

 

Small

3M 2F (2)

2M 1F (2)

3F (3)

10M 10F (6)

Missing

 

1M 1F (2)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Only abnormalities reported

Table 6 Summary Incidence of Daily Clinical Observations

                                                Day Numbers Relative to Start Date

Sex: Male

 

                                          Group 1               Group 2         Group 3         Group 4

                                        0(Control)         100 mg/kg bw/day 300 mg/kg bw/day 750 mg/kg bw/day

                                                                   

 

Scheduled kill                                                                                               

       Number of Animals                   12                    12              12              12     

       Days from ƒ to                    29 29                29 29          29 29          29 29   

                                                                                                             

Increased salivation                                                                                         

       Number of Animals                    .                     .               7              10     

       Days from ƒ to                       .                     .            15 28          15 27   

                                                                                                             

Noisy respiration                                                                                            

       Number of Animals                    .                     .               .               3     

       Days from ƒ to                       .                     .               .            11 27   

Littering                                                                                                    

       Number of Animals                    1                     .               1               2     

       Days from ƒ to                    38 38                   .            41 41          38 40   

                                                                                                             

Not Observed Due to Littering                                                                                

       Number of Animals                    1                     .               1               2     

       Days from ƒ to                    38 38                   .            41 41          38 40   

                                                                                                             

Scheduled kill                                                                                               

       Number of Animals                   12                    12              12              12     

       Days from ƒ to                    50 53                50 55          50 54          51 64   

                                                                                                             

Increased salivation                                                                                         

       Number of Animals                    .                     2               8              11     

       Days from ƒ to                       .                  47 48          26 51          11 60   

                                                                                                             

Noisy respiration                                                                                            

       Number of Animals                    .                     .               2               4     

       Days from ƒ to                       .                     .            39 48          29 63   

                                                                                                             

Generalised fur loss                                                                                         

       Number of Animals                    .                     .               1               .     

       Days from ƒ to                       .                     .            24 52             .     

Table 7 Group Mean Litter Values for Implantation Losses and Survival Indices

Group

 

Post -Implantation Loss (%)

Live Birth Index (%)

Viability Index 1 (%)

 

Viability Index 2 (%)

1

Mean

3.5

100.0

100.0

100.0

S.D.

5.9

0.0

0.0

0.0

N

9

9

9

9

 

 

 

 

 

 

2

Mean

4.6

100.0

93.3

100.0

S.D.

6.8

0.0

15.1

0.0

N

11

11

11

11

 

 

 

 

 

 

3

Mean

6.5

97.8

99.3

100.0

S.D.

8.1

4.9

2.3

0.0

N

10

10

10

10

 

 

 

 

 

 

4

Mean

5.6

100.0

95.5

98.9

S.D.

8.1

0.0

10.1

3.8

N

11

11

11

11

Dose Levels: Group 1 ƒ 0 (Control)   Group 2 ƒ 100 mg/kg bw/day   Group 3 ƒ 300 mg/kg bw/day   Group 4 ƒ 750 mg/kg bw/day            

Table 8 Group Mean Litter Values for Offspring Sex Ratio

Group

 

Sex Ratio (% Males)Post PartumDay:

At birth

1

4 BC

4 AC

7

13

1

Mean

58.3

58.3

58.3

61.5

61.5

61.5

S.D.

19.0

19.0

19.0

17.3

17.3

17.3

N

9

9

9

9

9

9

 

 

 

 

 

 

 

 

2

Mean

47.3

47.3

49.7

53.9

53.9

54.0

S.D.

12.4

12.4

16.5

15.2

15.2

15.0

N

11

11

11

11

11

11

 

 

 

 

 

 

 

 

3

Mean

49.1

48.5

48.2

53.4

53.4

53.4

S.D.

16.3

17.1

16.9

16.7

16.7

16.7

N

10

10

10

10

10

10

 

 

 

 

 

 

 

 

4

Mean

56.2

56.2

55.8

56.0

56.5

56.5

S.D.

14.5

14.5

12.9

11.8

11.1

11.1

N

11

11

11

11

11

11

Dose Levels: Group 1 ƒ 0 (Control)   Group 2 ƒ 100 mg/kg bw/day   Group 3 ƒ 300 mg/kg bw/day   Group 4 ƒ 750 mg/kg bw/day            

Table 9 Summary Incidence of Clinical Signs for Offspring

 

Dose Level (mg/kg bw/day)

 

0 (Control)

100

300

750

Number of litters ()

 

 

 

 

 

 

 

 

 

Birth to Day 4

 

 

 

 

Small

1F (1)

 

1M 1F (2)

1M 3F (2)

Found dead

 

1F (1)

2M (2)

1M 3F (2)

Missing

 

3M 7F (2)

2U (1)

1M (1)

Cold

 

 

1F (1)

4M 5F (3)

Bruise on snout

 

 

 

1M (1)

Weak

 

 

 

2F (2)

 

 

 

 

 

Day 5 to Day 7

 

 

 

 

Small

3M 2F (2)

2M 2F (2)

2F (2)

10M 11F (6)

Pale

1F (1)

 

 

 

Missing

 

 

 

1F (1)

 

 

 

 

 

Day 8 to Day 13

 

 

 

 

Small

3M 2F (2)

2M 1F (2)

3F (3)

10M 10F (6)

Missing

 

1M 1F (2)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Only abnormalities reported

Applicant's summary and conclusion

Conclusions:
The NOEL and NOAEL for reproduction and the survival and development of the offspring was considered to be 750 mg/kg bw/day. The NOEL and NOAEL for offspring growth was considered to be 300 mg/kg bw/day, due to lower body weight gain of the offspring at 750 mg/kg bw/day.