4,4'-(9H-fluoren-9-ylidene)bis(2-chloroaniline)

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October-December 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Also in accordance with GLP principles

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October-December 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Also in accordance with GLP principles.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

(1995)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3550 Reproduction/Developmental toxicity Screening test (2000)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 11 weeks
- Weight at study initiation: (P) Males: 269-299 g; Females: 192-225 g
- Fasting period before study: not applicable
- Housing: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (home cage) before mating. Females were caged together with males on a one-to-one basis in Marcolon plastic cages for mating. After mating, males were housed in their home cage, females were individually housed in Macrolon plastic cages. Pups were kept with the dam until termination.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 08 October To: 2 December 2013

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Remarks:

specific gravity 1.036

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made
for specific gravity of the vehicle. No correction was made for the purity/composition of the test substance.
Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at WIL Research Europe.

Details on mating procedure:

- M/F ratio per cage: 1/1 (one female was cohabitated with one male of the same treatment group, avoiding sibling mating).
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in vaginal lavage or by the appearance of an intravaginal copulatory plug referred to as Day 0.
- After successful mating each pregnant female was caged: see details on test animals and conditions

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase, according to a validated method. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Duration of treatment / exposure:

Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 41-55 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy). 2 Females of Group 1, one females of group 3 and one of group 4 were not dosed on Day 1 of lactation as these females were littering at the time of dosing. The omission of one day of dosing over a period of several weeks was not considered to affect the toxicological evaluation.

Frequency of treatment:

1/day, 7 d/wk

Details on study schedule:

- Age at mating of the mated animals in the study: Approximately 13 weeks

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on a 28-day oral toxicity study in which 0, 10, 100 and 1000 mg/kg bw/day were dosed by oral gavage to five rats/sex/group. At 1000 mg/kg bw/day, the following findings were noted: increased water consumption for females, reduced lymphocyte and total white blood cell counts for one female rat, higher adrenal and liver weights for females, and minimal centrilobular hepatocyte enlargement in the liver of one female. At 100 mg/kg bw/day, a slight increase in water consumption was recorded for females. No findings were noted at 10 mg/kg bw/day.
- Other: Based on these results, the liver and adrenals were weighed and collected during this project.

Parturition:
The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes and placentas cleaned up, nest build up and/or feeding of pups started). Females that were littering were left undisturbed.

Pups:
On Day 1 of lactation, all pups were individually identified by means of subcutaneous injection of Indian ink. Pups were not dosed directly but could have potentially be exposed to the test substance in utero, via maternal milk or from exposure to maternal urine/faeces.

Selection of animals for selected measurements:
5 animals/sex/dose were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination, organ weights and histopathology. Only females with live offspring were selected.

Positive control:

not applicable

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were made in all animals. The time of onset, grade and duration of any observed sign was recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION: Yes
- Time schedule: Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 postcoitum and on Days 1 and 4 of lactation.

WATER CONSUMPTION: Yes
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

OTHER: General reproduction data
Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
Spermatogenic staging profiles

Litter observations:

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

- Mortality/viability: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated. (Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7).)
- Clinical signs: At least once daily, detailed clinical observations were made for all animals.
- Body weights: Live pups were weighed on Days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation (by assessment of ano-genital distance).

GROSS EXAMINATION OF DEAD PUPS:
Yes, if necessary, defects or cause of death were evaluated.

Postmortem examinations (parental animals):

SACRIFICE and gross necropsy:
The animals were not deprived of food overnight. All animals surviving to the end of the observation period were deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

The numbers of former implantation sites and corpora lutea were recorded for all paired females.

HISTOPATHOLOGY/ORGAN WEIGHTS:
Adrenals, epididymides, liver and testes

HISTOPATHOLOGY:
According to the guidelines. Staging of spermatogenesis was examined for control and 1000 mg/kg bw/day dosed males and all males suspected to be infertile.

Postmortem examinations (offspring):

SACRIFICE
Pups surviving to planned termination were killed by decapitation on Days 5-6 of lactation. One pup (300 mg/kg bw/d group) was killed in extremis on Day 4 of lactation by decapitation.

GROSS NECROPSY
All pups were sexed and descriptions of all external abnormalities were recorded. At discretion of the Study Director, relevant abnormalities were collected and fixed in 10% buffered formalin; this was done for one pup from the mid dose group (300 mg/kg bw/day) that showed a wound on the left flank and had to be euthanized in extremis If possible, defects or cause of death were evaluated.

HISTOPATHOLOGY / ORGAN WEIGTHS
No

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Reproductive indices:

For each group, the following calculations were performed:
-Mating index (%): (Number of females mated/Number of females paired) x 100
-Fertility index (%): (Number of pregnant females/Number of females paired) x 100
-Conception index (%): (Number of pregnant females/Number of females mated) x 100
-Gestation index (%): (Number of females bearing live pups/Number of pregnant females) x 100
-Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

-Percentage live males at First Litter Check: (Number of live male pups at First Litter Check/Number of live pups at First Litter Check) x 100
-Percentage live females at First Litter Check: (Number of live female pups at First Litter Check/Number of live pups at First Litter Check) x 100
-Percentage of postnatal los: (Number of dead pups before planned necropsy/Number of live pups at First Litter Check) x 100
-Viability index: (Number of live pups before planned necropsy/Number of pups born alive) x 100

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment related clinical signs of toxicity were noted during the observation period. Incidental findings that were noted included scabbing, a wound, and alopecia. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.

Mortality:

no mortality observed

Description (incidence):

No mortality occured.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before or after allowance for body weight was similar between treated and control animals.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related microscopic findings.
There were four pairs of the 0 mg/kg bw/day group, two pairs of the 100 mg/kg bw/day group and two pairs of the 300 mg/kg bw/day group that failed to sire or deliver healthy pups. All findings recorded in the reproductive organs of these animals were within the normal range of background pathology. Spermatogenic staging profiles were normal for all males examined.
All microscopic findings recorded were considered to be within the normal range of background pathology encountered in rats of this age and strain.

Other effects:

not examined

Description (incidence and severity):

A minimum of eight pregnant females per group is advised by the OECD 421 test guideline. For the concurrent control group however, only six females were pregnant. These females also had nests with a mean sex ratio that fell outside the historical control data range. The small number of pregnant females, slightly high mean/median precoital time, slightly low number of corpora lutea and implantation sites, relatively small litter sizes, and the skewed sex ratio were attributed to chance for these control females. Having conducted over 86 repro screening studies in 2010-2013 (either alone, or in combination with 28-day repeated dose toxicity studies), WIL Research Europe has an extensive historical control database from animals of this same strain and supplier. As the data from the concurrent control group were not representative of the historical control data generated from this type of study, the data from the treated groups were also evaluated using the extensive historical control data available, and are included in the report. The extent and consistency of the historical control data allowed for a complete and thorough evaluation of the test substance on reproduction and development. All reproductive and developmental data from treated groups remained within the range of historical control data available.

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Spermatogenic staging profiles were normal for all males examined.

Reproductive performance:

no effects observed

Description (incidence and severity):

No toxicologically relevant effects on reproductive parameters were noted.
Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment. A total of eight mated females were not pregnant: four in the control group, two treated at 100 mg/kg bw/day and two treated at 300 mg/kg bw/day which resulted in decreased fertility and conception indices for the control group. This was considered a chance finding and not treatment related as it mostly concerned the control group and at 1000 mg/kg bw/day all females were pregnant. For a thorough evaluation of all reproduction data, historical control data were added to the report. The statistically significant increase for number of corpora lutea at 1000 mg/kg bw/day was not considered toxicologically relevant as all values were within normal limits, with the control group at the lower end.

Gestation
Gestation index was 100% for all groups and duration of gestation was similar among the groups.

Parturition/maternal care
No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed. However it might be postulated that one dam probably caused the wound on the flank of the pup. At this single occurrence, it was considered a chance finding.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed up to and including the highest dose tested.

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

One pup at 300 mg/kg bw/day showed an affected snout (missing skin and scabs) and lean or pale appearance. A body weight loss was noted for this animal (5.0 gram on Day 1 and 3.8 gram on Day 4). One pup at 1000 mg/kg bw/day showed indentation of the head. At this low incidence, these findings were considered to be of no toxicological relevance.

Mortality / viability:

no mortality observed

Description (incidence and severity):

Two pups at 100 mg/kg bw/day and two pups at 1000 mg/kg bw/day were missing during the first days of lactation; these pups were most likely cannibalised. No toxicological relevance was attributed to this since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
One pup at 300 mg/kg bw/day was killed in extremis on Day 4 of lactation. This pup showed a wound on the left flank. At this single occurrence at the mid dose group, it was not considered toxicologically relevant.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights of pups were considered to have been unaffected by treatment.

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed up to and including the highest dose tested.

Critical effects observed:

no

Reproductive effects observed:

no

Analysis of dose preparation.

- Accuracy of preparation: The concentrations analysed in the formulations of the doses of 100, 300 and 1000 mg/kg bw/day were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). No test substance was detected in the control formulation.

- Homogeneity: The formulations of the doses of 100 and 1000 mg/kg bw/day were homogeneous (i.e. coefficient of variation ≤ 10%).

- Stability: Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.

Conclusions:

In an oral OECD 421 rat reproduction/developmental screening study, no parental, reproduction or developmental effects were observed up to and including the highest dose tested. Therefore, the NOAEL is determined to be >=1000 mg/kg bw/day.

Executive summary:

The test substance was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 0, 100, 300 and 1000 mg/kg bw/day according to OECD 421. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 28 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 41-55 days). Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 6 hours at room temperature.

No parental toxicity was observed up to and including the highest dose level tested (1000 mg/kg bw/day). No treatment-related changes were noted in any of the parental parameters investigated in this study (i.e. clinical appearance, body weight, food consumption, macroscopic examination, organ weights, and microscopic examination). No reproduction toxicity nor any treatment related changes in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites) were observed

up to and including the highest dose level tested (1000 mg/kg bw/day).

No developmental toxicity nor any treatment related changes in any of the developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy) were observed up to and including the highest dose level tested.

Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg bw/day was derived.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test, July 1995.

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3550 Reproduction/Developmental toxicity Screening test (2000)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-(9H-fluoren-9-ylidene)bis(2-chloroaniline)

EC Number:

407-560-9

EC Name:

4,4'-(9H-fluoren-9-ylidene)bis(2-chloroaniline)

Cas Number:

107934-68-9

Molecular formula:

C25H18Cl2N2

IUPAC Name:

4-[9-(4-amino-3-chlorophenyl)-9H-fluoren-9-yl]-2-chloroaniline

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approx. 11 weeks
- Weight at study initiation: (P) Males: 269-299 g; Females: 192-225 g
- Fasting period before study: not applicable
- Housing: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (home cage) before mating. Females were caged together with males on a one-to-one basis in Marcolon plastic cages for mating. After mating, males were housed in their home cage, females were individually housed in Macrolon plastic cages. Pups were kept with the dam until termination.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 08 October To: 2 December 2013

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Remarks:

specific gravity 1.036

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made
for specific gravity of the vehicle. No correction was made for the purity/composition of the test substance.
Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at WIL Research Europe.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase, according to a validated method. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Details on mating procedure:

- M/F ratio per cage: 1/1 (one female was cohabitated with one male of the same treatment group, avoiding sibling mating.
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in vaginal lavage or by the appearance of an intravaginal copulatory plug referred to as Day 0.
- After successful mating each pregnant female was caged: see details on test animals and conditions

Duration of treatment / exposure:

Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 41-55 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy). 2 Females of Group 1, one females of group 3 and one of group 4 were not dosed on Day 1 of lactation as these females were littering at the time of dosing. The omission of one day of dosing over a period of several weeks was not considered to affect the toxicological evaluation.

Frequency of treatment:

1/d, 7d/wk

Duration of test:

See above

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on a 28-day oral toxicity study in which 0, 10, 100 and 1000 mg/kg bw/day were dosed by oral gavage to five rats/sex/group. At 1000 mg/kg bw/day, the following findings were noted: increased water consumption for females, reduced lymphocyte and total white blood cell counts for one female rat, higher adrenal and liver weights for females, and minimal centrilobular hepatocyte enlargement in the liver of one female. At 100 mg/kg bw/day, a slight increase in water consumption was recorded for females. No findings were noted at 10 mg/kg bw/day.
- Other: Based on these results, the liver and adrenals were weighed and collected during this project.

Parturition:
The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes and placentas cleaned up, nest build up and/or feeding of pups started). Females that were littering were left undisturbed.

Pups:
On Day 1 of lactation, all pups were individually identified by means of subcutaneous injection of Indian ink.
Pups were not dosed directly but could have potentially be exposed to the test substance in utero, via maternal milk or from exposure to maternal urine/faeces.

Selection of animals for selected measurements:
5 animals/sex/dose were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination, organ weights and histopathology. Only females with live offspring were selected.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were made in all animals. The time of onset, grade and duration of any observed sign was recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION: Yes
- Time schedule: Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 postcoitum and on Days 1 and 4 of lactation.

WATER CONSUMPTION: Yes
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

OTHER: General reproduction data
Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

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

Fetal examinations:

SACRIFICE
Pups surviving to planned termination were killed by decapitation on Days 5-6 of lactation. One pup (300 mg/kg bw/day group) was killed in extremis on Day 4 of lactation by decapitation.

GROSS NECROPSY
All pups were sexed and descriptions of all external abnormalities were recorded. At discretion of the Study Director, relevant abnormalities were collected and fixed in 10% buffered formalin; this was done for one pup from the mid dose group (300 mg/kg bw/day) that showed a wound on the left flank and had to be euthanized in extremis If possible, defects or cause of death were evaluated.

HISTOPATHOLOGY / ORGAN WEIGTHS
No

The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross abnomalies, weight gain, physical or behavioural abnormalities.
- Mortality: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made in all animals.
- Body weights: Live pups were weighed on Days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance).

GROSS EXAMINATION OF DEAD PUPS: Yes
If possible, defects or cause of death were evaluated.

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Indices:

Reproductive indices; For each group, the following calculations were performed:
- Mating index: Number of females mated/Number of females paired x 100
- Fertility index: Number of pregnant females/Number of females paired x 100
- Conception index: Number of pregnant females/Number of females mated x 100
- Gestation index: Number of females bearing live pups/Number of pregnant females x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring indices:
- Percentage live males at First Litter Check: (Number of live male pups at First Litter Check/Number of live pups at First Litter Check) x 100
- Percentage live females at First Litter Check: (Number of live female pups at First Litter Check/Number of live pups at First Litter Check) x 100
- Percentage of postnatal loss Days 0-4 of lactation: (Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check) x 100
- Viability index: (Number of live pups on Day 4 of lactation / Number of pups born alive) x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related clinical signs of toxicity were noted during the observation period. Incidental findings that were noted included scabbing, a wound, and alopecia. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed
and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.

Mortality:

no mortality observed

Description (incidence):

No mortality occured.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before or after allowance for body weight was similar between treated and control animals.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes were noted in organ weights and organ to body weight ratios.
The statistically significant increase noted for liver weights and liver to body weight ratios for females treated at 1000 mg/kg bw/day were not considered toxicologically relevant as this change was due to their pregnancy status (there were four non-pregnant animals in the control group, two in both the low and mid dose groups and none in the high dose group). The mean liver weights were calculated for 10 females per group (pregnant and non-pregnant). When looking at the individual values for female liver weights, the non-pregnant females have a lower weight (which is normal for their physiological state). When excluding these non-pregnant values, the mean values for liver weights are 10.34, 10.84, 10.61 and 11.00 gram and for liver to body weight ratios are 3.85%, 4.11%, 3.94% and 4.09% for dosed groups of 0, 100, 300 and 1000 mg/kg bw/day, respectively.
The statistically significantly change noted for relative liver weight for males treated at 100 mg/kg bw/day was not considered toxicologically relevant as no dose response was noted and all values were within normal limits.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.
The incidence of incidental findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose related incidence trend. These necropsy findings were therefore considered to be of no toxicological
relevance, and included pelvic dilation of the kidneys, nodule at the epididymides, scabbing, uterus containing fluid, focus on the clitoral glands or thymus, alopecia, enlarged adrenals or clitoral glands, diaphragmatic hernia of the liver, and discolouration of the clitoral glands.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related microscopic findings.
There were four pairs of controls, two pairs of 100 mg/kg bw/day dosed group and two pairs of the 300 mg/kg bw/day dosed group that failed to sire or deliver healthy pups. All findings recorded in the reproductive organs of these animals were within the normal range of background pathology. Spermatogenic staging profiles were normal for all males examined.
All microscopic findings recorded were considered to be within the normal range of background pathology encountered in rats of this age and strain.

Maternal developmental toxicity

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Gestation index was 100% for all groups and duration of gestation was similar among the groups.

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

A total of eight mated females were not pregnant: four in the control group, two treated at 100 mg/kg bw/day and two treated at 300 mg/kg bw/day which resulted in decreased fertility and conception indices for the control group. This was considered a chance finding and not treatment related as it mostly concerned the control group and at 1000 mg/kg bw/day all females were pregnant.

A minimum of eight pregnant females per group is advised by the OECD 421 test guideline. For the concurrent control group however, only six females were pregnant. These females also had nests with a mean sex ratio that fell outside the historical control data range. The small number of pregnant
females, slightly high mean/median precoital time, slightly low number of corpora lutea and implantation sites, relatively small litter sizes, and the skewed sex ratio were attributed to chance for these control females. Having conducted over 86 repro screening studies in 2010-2013 (either alone,
or in combination with 28-day repeated dose toxicity studies), WIL Research Europe has an extensive historical control database from animals of this same strain and supplier. As the data from the concurrent control group were not representative of the historical control data generated from this type of study, the data from the treated groups were also evaluated using the extensive historical control data available, and are included in the report. The extent and consistency of the historical control data allowed for a complete and thorough evaluation of the test substance on reproduction and
development. All reproductive and developmental data from treated groups remained within the range of historical control data available.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant effects on reproductive parameters were noted.
Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment. The statistically significant increase for number of corpora lutea at 1000 mg/kg bw/day was not considered toxicologically relevant as all values were within normal limits, with the control group at the lower end.

Parturition/maternal care
No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed. However it might be postulated that one dam probably caused the wound on the flank of the pup. At this single occurrence, it was considered a chance finding.

Details on maternal toxic effects:

No maternal toxicity was noted.
No toxicologically relevant effects on reproductive parameters were noted.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights of pups were considered to have been unaffected by treatment.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in postnatal survival:

effects observed, non-treatment-related

Description (incidence and severity):

Two pups at 100 mg/kg and two pups at 1000 mg/kg bw/day were missing during the first days of lactation; these pups were most likely cannibalised. No toxicological relevance was attributed to this since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
One pup at 300 mg/kg bw/day was killed in extremis on Day 4 of lactation. This pup showed a wound on the left flank. At this single occurrence at the mid dose group, it was not considered toxicologically relevant.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One pup at 300 mg/kg bw/day showed an affected snout (missing skin and scabs) and lean or pale appearance. A body weight loss was noted for this animal (5.0 gram on Day 1 and 3.8 gram on Day 4). One pup at 1000 mg/kg showed indentation of the head. At this low incidence, these findings were considered to be of no toxicological relevance.

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects: no effects.
Remark: Only early postnatal pup development parameters were examined including body weight, post-natal loss, sex ratio, clinical signs, body weight and external macroscopy.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: developmental toxicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

In an oral OECD 421 screening study with rats, the parental and developmental NOAEL were both determined to be >=1000 mg/kg bw/day.

Executive summary:

The test substance was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 0, 100, 300 and 1000 mg/kg bw/day according to OECD 421 and following GLP principles.

Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 28 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 41-55 days). Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 6 hours at room temperature.

No parental toxicity was observed up to and including the highest dose level tested (1000 mg/kg bw/day). No treatment-related changes were noted in any of the parental parameters investigated in this study (i.e. clinical appearance, body weight, food consumption, macroscopic examination, organ weights, and microscopic examination). No reproduction toxicity nor any treatment related changes in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites) were observed up to and including the highest dose level tested (1000 mg/kg bw/day).

No developmental toxicity nor any treatment related changes in any of the developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy) were observed up to and including the highest dose level tested.

Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg bw/day was derived.