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Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

Information on reproductive toxicity is available from an OECD 422, as well as an OECD 443 study.


 


OECD 422:Combined 28-day repeated dose toxicity study with the reproductin/developmental toxicity screening test in rats by oral gavage:


Dosages: 25, 75, 225 mg/kg bw/d


Parental NOAEL: 25 mg/kg bw/day:


Hepatic toxicity in animals of both sexes at 225 mg/kg bw/day. At 75 mg/kg bw/d, morphologic liver findings were regarded to be an adaptive, non-adverse response. However, additional changes at 75 mg/kg bw/d in clinical biochemistry and a relative liver weight increase were considered toxicologically relevant.


 


Reproduction NOAEL: 225 mg/kg bw/day:


No reproductive toxicity was observed up to 225 mg/kg bw/day.


 


OECD 443: Extended One-Generation Reproduction Toxicity Study in Wistar Rats Administration via the Diet:


Dosages: 200, 600, 1800 ppm. With 10 weeks premating, extension of cohort 1B and developmental neurotoxicity cohorts 2A and 2B.


General, systemic toxicity LOAEL F0: approx. 18 mg/kg bw/d (200 ppm):


Evidence for liver toxicity and corresponding thyroid histopathology and thyroid hormone changes in all test groups


 


General, systemic toxicity NOAEL F1: approx. 18 mg/kg bw/d (200ppm):


Distinct toxicity such as decreased body weight/body weight gain, anemia as well as liver and thyroid toxicity.


 


Fertility and reproduction NOAEL in F0 and F1: Approx. 54 mg/kg bw/d (600ppm):


Lower numbers of implants and subsequently smaller litters.


 


 


 

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and OECD guideline compliant study with well-characterized test material
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted in 1996 (This version does not include the endocrine disruptor parameters introduced with the update of 28 July 2015)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): Benzenamine, N-phenyl-,reaction products with 2,4,4-trimethylpentene
- Substance type: UVCB
- Physical state: Clear slightly yellow viscous liquid
- Analytical purity: 100% UVCB
- Purity test date: 2014 , study number 13L00223
- Lot/batch No.: 40401913D
- Expiration date of the lot/batch: 09 February 2016
- Stability under test conditions: stable
- Storage condition of test material: At room temperature in the dark

Test substance handling: Avoid temperatures <10°C, maximum temperature:30°C. In the case of crystallization, heat up the substance (temperature up to 30°C) until a clear solution is obtained
Specific Gravity / Density 0.975 g/cm3 (20°C)
pH 5.1-6.2 (1%(m), 20-25°C) (as suspension)
Stability at higher temperatures Maximum temperature: 30°C
Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 10-12 weeks
- Weight at study initiation: 353 g (males), 210 g (females)
- Fasting period before study: none
- Housing: groups of 5 animals/sex/cage (premating males and females and postmating males), otherwise single cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%,
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 13 March 2014 To: 1 May 2014
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) are be prepared daily within 6 hours prior to dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Test item is insoluble in water, but soluble in corn oil
- Amount of vehicle (if gavage): 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.


Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal
copulatory plug. This day was designated Day 0 post-coitum.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
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 90-110% 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:
28 days (males), ca 53 days (females)


Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
25, 75 and 225 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Range-finding standy
- Rationale for animal assignment (if not random): Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: mortality, clinical observations

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the first exposure and at the end of the study as part of the functional observation battery (FOB)

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of male and female animals were recorded shortly before the start of
administration of the test item at randomization and at the start of the study (day 0). Males
were weighed weekly until sacrifice. Females were weighed once per week during the premating period. Mated females were
weighed on days 0, 7, 14 and 21 during presumed gestation and on day 0 and 4 of lactation.
Non-mated females were weighed once per week after the mating period. In addition, the
animals were weighed on their scheduled necropsy date in order to calculate the correct organ to body weight ratios.


FOOD CONSUMPTION:
Feed consumption was measured per cage over weekly intervals during the study, with exemption of the mating period, during which no feed consumption was registered.


HAEMATOLOGY
Prior to sacrifice, 5 animals/sex/group were fasted overnight (water was freely available) and blood was taken from the aorta during necropsy, whilst under CO2/O2 anaesthesia. K3-EDTA was used as anticoagulant. In each sample the following determinations were carried out:
haemoglobin
packed cell volume
red blood cell count
reticulocytes
total white blood cell count
differential white blood cell counts (neutrophils, lymphocytes, eosinophils,
basophils, monocytes)
prothrombin time
thrombocyte count
mean corpuscular volume (MCV; calculated)
mean corpuscular haemoglobin (MCH; calculated)
mean corpuscular haemoglobin concentration (MCHC; calculated)
activated partial prothoplastin time (APTT)
red blood cell districution width (RDW)

CLINICAL CHEMISTRY
Prior to sacrifice, 5 animals/sex/group were fasted overnight (water was freely available) and blood was taken from the aorta during necropsy, whilst CO2/O2 anaesthesia. Blood was collected in heparinized plastic tubes and plasma was prepared by centrifugation. The following measurements were made in the plasma:
alkaline phosphatase activity (ALP),
aspartate aminotransferase activity (ASAT),
alanine aminotransferase activity (ALAT),
gamma glutamyl transferase activity (GGT),
total protein,
albumin,
ratio albumin to globulin (calculated),
urea,
creatinine,
glucose (fasting),
bilirubin (total),
cholesterol (total),
triglycerides,
phospholipids,
calcium (Ca),
sodium (Na),
potassium (K),
chloride (Cl),
inorganic phosphate (PO4),
bile acids


Thyroid hormone analyses:
Thyroid stimulating hormone (TSH) μIU/mL
total triiodothyronine (T3) ng/dL
total thyroxine (T4) μg/dL


NEURO-BEHAVIOURAL TESTING (FOB) AND SPONTANEOUS MOTOR ACTIVITY
During neuro-behavioural testing, the observer was unaware of the treatment of the animals. FOB and spontaneous motor activity were assessed in all study animals during the predose phase and in 5 animals/sex/group at the end of the study.


Sacrifice and pathology
GROSS NECROPSY AND HISTOLOGY OF PARENTAL ANIMALS
All surviving male and female parent animals were sacrificed by exsanguination from the abdominal aorta whilst under CO2/O2 anaesthesia at necropsy and then examined grossly for pathological changes. Male animals were sacrificed after the mating period. Female animals were sacrificed at or shortly after day 4 of lactation. A necropsy was performed on animals that died intercurrently (if not precluded by autolysis) or that had to be killed because they were moribund. Prior to preservation of organs/tissues, the following organ weights were recorded: adrenals, brain, heart, kidneys, liver, ovaries, prostate, seminal vesicles, spleen, thymus, thyroid, uterus. Samples of the following tissues and organs of all parent animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde; except for the testes which were preserved in Bouin's fixative:

- ovaries (after counting the corpora lutea)
- uterus (after counting of the implantation sites)
- testes
- epididymides
- seminal vesicles
- prostate
- all gross lesions

In addition of 5 animals/sex/group following organs were preserved:
adrenals
bone marrow (femur)
brain (including sections of cerebrum, cerebellum, medulla/pons)
caecum
cervix
clitorial gland
colon
coagulation gland
duodenum
eyes
femur including joint
heart
ileum
jejunum (including Peyer's patches)
kidneys
liver
lungs
mesenterial and axillary lymph nodes
ovaries
peripheral nerve (sciatic or tibial)
pituitary gland
preputial gland
prostate
rectum
seminal vesicles (including coagulation gland)
skeletal muscle (thigh)
spinal cord (cervical, mid-thoracic and lumbar)
spleen
stomach*
thymus
thyroid (including parathyroid)
trachea
urinary bladder
uterus
vagina
* Non glandular (“forestomach”) and glandular (fundus, pylorus) parts were examined
microscopically.

Microscopic examination was performed on the collected organs of all animals of the control (group 1) and high dose group (group 4). If treatment-related changes were observed in the high-dose group, the evaluation of these tissues/organs was extended to the intermediate-dose groups (2 and 3).
In addition, reproductive organs of males that failed to sire (did not mate or mated females were not pregnant) and females that were non-mated or non-pregnant, of the low- and mid-dose groups, were microscopically examined. Furthermore, organs showing gross lesions of animals of all groups were microscopically examined.


Sperm parameters (parental animals):
Parameters examined in male parental generations:
testis weight, epididymis weight, spermatogenesis.
Slides of the testes were made of all males of Groups 1 and 4 and stained with PAS/haematoxylin to assess spermatogenesis.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals [day 28, details see entries for repeated dose toxicity]
- Maternal animals: All surviving animals [between postnatal days 4 and 7]
GROSS NECROPSY AND HISTOLOGY OF PARENTAL ANIMALS
All surviving male and female parent animals were sacrificed by exsanguination from the abdominal aorta whilst under CO2/O2 anaesthesia at necropsy and then examined grossly for pathological changes. Male animals were sacrificed after the mating period. Female animals were sacrificed at or shortly after day 4 of lactation. A necropsy was performed on animals that died intercurrently (if not precluded by autolysis) or that had to be killed because they were moribund. Prior to preservation of organs/tissues, the following organ weights were recorded: adrenals, brain, heart, kidneys, liver, ovaries, prostate, seminal vesicles, spleen, thymus, thyroid, uterus. Samples of the following tissues and organs of all parent animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde; except for the testes which were preserved in Bouin's fixative:

- ovaries (after counting the corpora lutea)
- uterus (after counting of the implantation sites)
- testes
- epididymides
- seminal vesicles
- prostate
- all gross lesions
In addition of 5 animals/sex/group following organs were preserved:
adrenals
bone marrow (femur)
brain (including sections of cerebrum, cerebellum, medulla/pons)
caecum
cervix
clitorial gland
colon
coagulation gland
duodenum
eyes
femur including joint
heart
ileum
jejunum (including Peyer's patches)
kidneys
liver
lungs
mesenterial and axillary lymph nodes
ovaries
peripheral nerve (sciatic or tibial)
pituitary gland
preputial gland
prostate
rectum
seminal vesicles (including coagulation gland)
skeletal muscle (thigh)
spinal cord (cervical, mid-thoracic and lumbar)
spleen
stomach*
thymus
thyroid (including parathyroid)
trachea
urinary bladder
uterus
vagina
* Non glandular (“forestomach”) and glandular (fundus, pylorus) parts were examined
microscopically.

Microscopic examination was performed on the collected organs of all animals of the control (group 1) and high dose group (group 4). If treatment-related changes were observed in the high-dose group, the evaluation of these tissues/organs was extended to the intermediate-dose groups (2 and 3).
In addition, reproductive organs of males that failed to sire (did not mate or mated females were not pregnant) and females that were non-mated or non-pregnant, of the low- and mid-dose groups, were microscopically examined. Furthermore, organs showing gross lesions of animals of all groups were microscopically examined.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 5- 7 days of age.
- These animals were subjected to postmortem examinations (macroscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.]

Descriptions of all external abnormalities were recorded. The stomach of pups not surviving to the scheduled necropsy date were examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

Statistics:
Statistics
If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref. 2; many-toone t-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 (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Reproductive indices:
Mating index (%) Number of females mated x 100 / Number of females paired

Fertility index (%) Number of pregnant females x 100 / Number of females paired

Conception index (%) Number of pregnant females x 100 / Number of females mated

Gestation index (%) Number of females bearing live pups x 100 / Number of pregnant females

Duration of gestation Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
Percentage live females at first litter check: Number of live female pups at First Litter Check x 100 Number of live pups at First Litter
Check

Percentage live males at first litter check: Number of live male pups at First Litter Check x 100 / Number of live pups at First Litter Check

Percentage of postnatal loss: Number of dead pups before planned necropsy x 100 / Number of live pups at First Litter Check

Viability index: Number of live pups before planned necropsy x 100 / Number of pups born alive
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
liver
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and spermatogenic staging profiles were normal for all males examined.

No clinical signs of toxicity were noted during the observation period.
Salivation was seen after dosing for animals at 75 and 225 mg/kg bw/day. This was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to possible irritancy/taste of the test substance. Alopecia was seen for one female at 75 mg/kg bw/day. This was the only other clinical sign noted and
was incidental in nature.No toxicologically relevant effects on reproductive parameters were noted with treatment up to 225 mg/kg bw/day.

The mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment. Actual numbers are provided in the table below.
There were 10, 10, 10 and 9 pregnant females in the control, 25, 75 and 225 mg/kg bw/day groups, respectively.
For female nos. 46 (Group 1) and 55 (Group 2), the number of pups were slightly higher than the number of implantations. This was considered caused by normal resorption of these areas as these enumerations were performed on Day 5 or 7 of lactation.

Dose descriptor:
NOEL
Remarks:
fertility
Effect level:
>= 225 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects on fertility observed at the highest tested dose.
Dose descriptor:
NOAEL
Remarks:
parental toxicity
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: adverse effects on liver at 75 and 225 mg/kg bw
Critical effects observed:
yes
Lowest effective dose / conc.:
25 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Gestation
The gestation index and duration of gestation were similar between all groups.

Parturition/maternal care
Female no. 50 (Group 1) was euthanized in extremis after having a prolonged parturition. No other 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 and
no deficiencies in maternal care were observed.

Early postnatal pup development
The number of dead pups at first litter check and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings. At 225 mg/kg bw/day the mean number of living pups at first litter check (7.9) was significantly lower
than for controls (10.9). Female no. 80 had only 4 pups, which contributed to this slightly low mean. Discounting her data, there were a mean of 8.4 pups/litter, which was also lower than controls and an effect of treatment could not be excluded.
The postnatal loss was also significantly higher for animals at 225 mg/kg bw/day with 11.3% of living pups lost (8 pups over 3 litters) than controls (no pups lost). High dose animals also had a correspondingly low viability index of 88.7%. Mortality
One pup was found dead at the first litter check and one pup was missing on postnatal Day 2 at 25 mg/kg bw/day. At 225 mg/kg bw/day, 7 pups (4 females, 3 males) went missing between Days 2 and 5 of lactation and 1 female pup was euthanized in extremis on lactation Day 1. Missing pups were most the missing pups at 225 mg/kg bw/day were from a single litter (no. 75; three other pups from this litter survived to the end of the necropsy period), two were from another litter (no. 74). The majority of the pups came from a single litter and there were no signs of ill health in other pups from this dose level. However, when taken together with the lower mean number of pups at first litter check, an effect of treatment could not be excluded. No toxicological relevance was attributed to the dead/missing pups at 25 mg/kg bw/day since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Clinical signs
Clinical signs noted for pups who went missing, died or were euthanized in extremis included cold, no
milk in the stomach, wounds (various body areas), pale and lean appearance. Incidental clinical signs
noted for pups surviving to the scheduled necropsy period included scabs (various body areas) and
pale appearance. The nature and incidence of these signs are not uncommon for pups of this age and
strain and were not considered to be treatment related or toxicologically relevant.

Body weights
Body weights of pups were unaffected by treatment up to 225 mg/kg bw/day.

Macroscopy
Sore on the throat region was seen for the pup that was euthanized in extremis. This was the only
macroscopic finding seen for pups that died before the scheduled necropsy. Scabbing on the chest
was the only finding noted for pups surviving to the scheduled necropsy. These findings were both
incidental in nature.
Dose descriptor:
NOEL
Generation:
F1
Effect level:
75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Increase in postnatal pup mortality at 225 mg/kg bw., mostly in one animal and unlikely to be a true effect
Critical effects observed:
not specified
Reproductive effects observed:
yes
Lowest effective dose / conc.:
225 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes

No toxicologically relevant effects on reproductive parameters were noted with treatment up to 225 mg/kg bw/day. The mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.

There were 10, 10, 10 and 9 pregnant females in the control, 25, 75 and 225 mg/kg bw/day groups, respectively. For female nos. 46 (Group 1) and 55 (Group 2), the number of pups were slightly higher than the number of implantations. This was considered caused by normal resorption of these areas as these enumerations were performed on Day 5 or 7 of lactation.

No toxicologically relevant effects on the gestation index and duration, parturition, maternal care or on most aspects of early postnatal pup development (clinical signs, body weight and macroscopy) were observed up to 225 mg/kg bw/day.

GROUP 1 CONTROL GROUP 2
25 MG/KG
GROUP 3
75 MG/KG
GROUP 4
225 MG/KG
Females paired 10 10 10 10
Females mated 10 10 10 10
Pregnant females 10 10 10 9
Non-pregnant female 0 0 0 1
Females killed in extremis 1 0 0 0
Females with living pups on Day 1 9 10 10 9
Mating index (%) 100 100 100 100
(Females mated / Females paired) * 100
Fertility index (%) 100 100 100 90
(Pregnant females / Females paired) * 100
Conception index (%) 100 100 100 90
(Pregnant females / Females mated) * 100
Gestation index (%) 90 100 100 100
(Females with living pups on Day 1 / Pregnant females) * 100

control 25 mg/kg  75 mg/kg  225 MG/KG
NECROPSY
Corpora Lutea
MEAN 13.2 13.7 12.1 12.2
ST.DEV 2.3 3.7 2.0 2.0
N 10 10 10 9
Implantations MEAN 10.9 11.5 10.5 9.2
ST.DEV 2.4 2.2 1.7 2.3
N 10 10 10 9

+/++ Steel-test significant at 5% (+) or 1% (++) level

  control 25 mg/kg bw 75 mg/kg bw 225 mg/kg bw
Total 9 10 10 9
DURATION OF GESTATION        
Days, MEAN (+) 21.3 21.2 21.1 21.1
ST.DEV. 0.5 0.6 0.6 0.8
N 9 10 10 9
DEAD PUPS AT FIRST LITTER CHECK        
LITTERS AFFECTED (#) 0 1 0 0
TOTAL no of dead pups 0 1 0 0
MEAN (+) 0 0.1 0 0
ST.DEV. 0 0.3 0 0
N 9 10 10 9
LIVING PUPS AT FIRST LITTER CHECK        
% of MALES / FEMALES )# 57/43 55/45 51/49 45/55
TOTAL   98 105 94 71
MEAN (+) 10.9 10.5 9.4 7.9+
ST.DEV. 2.6 2.5 2 1.8
N 9 10 10 9
Postnatal loss        
% of living pups 0 1 0 11.3
LITTERS AFFECTED (#) 0 1 0 3
Total no of pups lost (#) 0 1 0 8##
MEAN (+) 0 0.1 0 0.9
ST.DEV. 0 0.3 0 1.7
N 9 10 10 9
Viability Index (#) 100 99 100 88.7#

+/++ Steel-test significant at 5% (+) or 1% (++) level

Viability index = (Number of alive pups before planned necropsy / Number of pups born alive) *100

# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level

Number of pups at first litter check and postnatal loss for the high dose group (225 mg/kg bw)

Female no. Days of Gestation Dead males at first litter check Dead females at first litter check Living males at first litter check Living females at first litter check Living pups (total) Postnatal loss (males) Postnatal loss (females)
72 22 0 0 5 5 10 0 0
73 20 0 0 2 5 7 0 0
74 21 0 0 6 2 8 1 1
75 21 0 0 2 6 8 2 3
76 22 0 0 5 3 8 0 0
77 21 0 0 3 5 8 0 1
78 20 0 0 2 6 8 0 0
79 21 0 0 7 3 10 0 0
80 22 0 0 0 4 4 0 0
TOTAL   0 0 32 39 71 3 5
N 9 9 9 9 9 9 9 9
MEAN 21.1 0.0 0.0 3.6 4.3 7.9 0.3 0.6
ST.DEV. 0.8 0.0 0.0 2.3 1.4 1.8 0.7 1.0

Pup Bodyweights (g) during lactation

control 25 mg/kg bw 75 mg/kg bw 225 mg/kg bw
Postnatal Day 1 M MEAN 6.4 6.3 6.4 6.1
ST.DEV. 0.8 0.5 0.5 0.8
N 9 10 10 8
F MEAN 6.2 5.9 6.1 5.9
ST.DEV. 0.8 0.6 0.6 0.9
N 9 9 10 9
M+F MEAN 6.3 6.1 6.3 6.0
ST.DEV. 0.7 0.6 0.5 0.8
N 9 10 10 9
Postnatal Day 4 M MEAN 9.5 9.8 9.7 8.7
ST.DEV. 1.5 1.1 1.0 2.2
N 9 10 10 8
F MEAN 9.3 9.2 9.3 9.0
ST.DEV. 1.4 1.4 1.2 1.4
N 9 9 10 9
M+F MEAN 9.4 9.6 9.5 9.0
ST.DEV. 1.4 1.2 1.1 1.6
N 9 10 10 9

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Endpoint:
extended one-generation reproductive toxicity - with F2 generation and developmental neurotoxicity (Cohorts 1A, 1B with extension, 2A and 2B)
Remarks:
Ten weeks premating exposure duration for the parental (P0) generation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14.Oct.2019 - 26.May.2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
25.Jun.2018
Cohorts 1A, 1B (extended), 2A, 2B (Developmental neurotoxicity)
GLP compliance:
yes (incl. QA statement)
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS [please address all points below]:

- Premating exposure duration for parental (P0) animals: 10 weeks premating as requested by ECHA
- Basis for dose level selection: Doses were selected based on a dose range finding study performed prior to this study
- Inclusion of extension of Cohort 1B as requested by ECHA
- Termination time for F2: At weaning
- Inclusion of developmental neurotoxicity Cohorts 2A and 2B as requested by ECHA
- Exclusion of developmental immunotoxicity Cohort 3, no indications of immunotoxicity
- Route of administration: oral via the feed
Specific details on test material used for the study:
- Content: 100g/100 g UVCB
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on species / strain selection:
The rat is the preferred animal species for reproduction studies according to test guidelines.
This strain was selected since extensive historical control data were available for Wistar rats.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 28 (+/-1) days
- Fasting period before study: Not specified
- Housing: From delivery to randomization and during the study period, the rats were housed together (up to 5 animals per sex and cage) in Polysulfonate cages Typ 2000P (H-Temp) with the following exceptions:
• During overnight matings (male and female mating partners were housed together), gestation, lactation, females after weaning and for FOB and MA the animals were housed individually in Polycarbonate cages type III.
• Dams and their litters were housed together until PND 21/22 in Polycarbonate cages type III.
- Diet: Mouse and rat maintenance diet “GLP”, supplied by Garanovit AG,
Kaiseraugst, Switzerland ad libitum
- Water: Drinking water was supplied from water
bottles (ad libitum)
- Acclimation period: Approx. 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 45-65%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: To: 15.Oct.2019 - 07.Jul.2020
Route of administration:
oral: feed
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required quantity of test substance was weighed in a beaker depending on the dose group and thoroughly mixed with a small amount of food. Then further amounts of food were added to this premix and thoroughly mixed for 3 minutes. Afterwards, further amounts of food, depending on the dose group, were added to this premix in order to obtain the desired concentrations. Mixing of this final mix was carried out for about 10 minutes in a laboratory mixer.

DIET PREPARATION
- Rate of preparation of diet (frequency): Not specified
- Mixing appropriate amounts with (Type of food): Mouse and rat maintenance diet “GLP”, supplied by Garanovit AG, Kaiseraugst, Switzerland ad libitum

Details on mating procedure:
- M/F ratio per cage: 1 : 1
- Length of cohabitation: Overnight
- Proof of pregnancy: Sperm in vaginal smear referred to as day 0 pregnancy
- After 2 weeks of unsuccessful pairing replacement of first male by another male from the same dose group
- After successful mating each pregnant female was caged (how): Animals were housed individually in Polycarbonate cages type III.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analyses of the test substance preparations were carried out as a separate study, under the responsibility of a Study Director of the respective test facility. This study was carried out in compliance with the Principles of Good Laboratory Practice.
Analytical verifications of the stability of the test substance in the diet for a period of 35 days at room temperature were performed before the start of the study.
Analyses were carried out 5 times during the study. At the beginning, in the middle and toward the end of the study, as well as for female diets once in each lactation period:
Concentration control (mid dose) and Homogeneity analyses (low + high dose).

Identity was verified by IR and NMR spectroscopy. Individual components were identified via HPLC mass spectroscopy fingerprinting.

The stability of test substance in rat diet was demonstrated for a period of 35 days at room temperature. The homogeneity of the mixtures was verified and the mean of mean recovery rates of all investigated samples of Benzenamine, N-phenyl-, reaction products with 2,4,4-trimethylpentene in the diet were in the expected range of the target concentrations (90 - 110%), demonstrating the correctness of the preparations.
Duration of treatment / exposure:
All animals were exposed continuously throughout the study with a 10 week premating period for P0 animals.

During the lactation period the test substance concentrations in the diet of the F0 and F1 females were reduced to 50%

F0: 17 weeks
F1 1A: 13 weeks
F1 1B: 19 - 25 weeks
F1 2A: 11 weeks
F1 2B: 3 weeks
F2: 3 weeks
Frequency of treatment:
Continuously via the feed
Details on study schedule:
- F1 parental animals not mated until 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: Approx. 13 weeks
Dose / conc.:
0 ppm
Remarks:
Control group
Dose / conc.:
200 ppm
Remarks:
Low dose group; Approx. 18 mg/kg bw/d
Dose / conc.:
600 ppm
Remarks:
Intermediate dose group; Approx. 54 mg/kg bw/d
Dose / conc.:
1 800 ppm
Remarks:
High dose group; Approx. 167 mg/kg bw/d
No. of animals per sex per dose:
F0 generation: 25
F1 generation:
Cohort 1A: 20
Cohort 1B: 25
Cohort 2A: 10
Cohort 2B: 10
PND 4-pups (F1 + F2): 10
PND 22-pups (F1 + F2): 10
Control animals:
yes
Details on study design:
- Dose selection rationale: Doses were selected based on a range finding study
- Rationale for animal assignment: The assignment of the animals to the different test groups was carried out using a randomization program, according to their weight one day before the beginning of the administration period (day -1).
- Fasting period before blood sampling for clinical biochemistry: 16h
Positive control:
No
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly
- Parameters in table 1 were checked

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly; During gestation and lactation F0 and F1B females were weighed on GD 0, 7, 14 and 20 and on PND 1, 4, 7, 10, 14, 18 and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:

OTHER:
Clinical Pathology in F0 parental and cohort 1A animals
Samples were withdrawn from 10 F0 parental and cohort 1A males and females per group at termination. Blood samples were taken from animals by puncturing the retrobulbar venous plexus following isoflurane anesthesia. Blood sampling and blood examinations were carried out in a randomized sequence. The list of randomization instructions was compiled with a computer. In the afternoon preceding the day of urinalysis, the animals were individually transferred into
metabolism cages (no food or drinking water provided); on the following morning, the individual urine specimens were examined in a randomized sequence (the list of randomization instructions was compiled with a computer). The assays of blood and serum parameters were performed under internal laboratory quality
control conditions with reference controls to assure reliable test results.

The concentrations of TSH and T4 were determined.

Haematology parameters in table 6 were analyzed.
Clinical chemistry parameters in table 7 were analyzed.
Urinalysis parameters in table 8 were analyzed.
Oestrous cyclicity (parental animals):
Estrous cycle data were evaluated for F0 and F1B females over a 3 week period prior to mating until evidence of mating occurred. In all cohort 1A females, vaginal smears were collected after vaginal opening until the first cornified smear (estrous) was recorded. The estrous cycle also was evaluated in cohort 1A females for 2 weeks around PND 75. Moreover, the estrous stage of each F0, 1A and 1B female was determined on the day of scheduled sacrifice.
Sperm parameters (parental animals):
Parameters examined in P/F1 male parental generations:
Various sperm parameters (motility, sperm head count (testis and cauda epididymides), morphology) were assessed in the F0 generation males and cohort 1A males at scheduled sacrifice or after appropriate staining
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter); All culled pups were subjected to a macroscopic (external and visceral) examination.

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring:
number and sex of pups, sex ratio, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), anogenital index, pup weight on the day of AGD, presence of nipples/areolae in male pups, pup organ weights, vaginal opening, preputial separation

GROSS EXAMINATION OF DEAD PUPS:
yes
On PND 4, as a result of standardization, selected F1 pups were sacrificed by decapitation under isoflurane anesthesia and blood was sampled for determination of Total thyroxine (T4) and Thyroid stimulating hormone (TSH).

After standardization on PND 4, the surplus F2 pups were sacrificed under isoflurane anesthesia with CO2. After sacrifice, these pups were examined externally, eviscerated and their organs were assessed macroscopically

On PND 22, the surplus F1 generation pups that were not used for the formation of the cohorts or any investigations were sacrificed under isoflurane anesthesia with CO2 and were examined in the pathology lab. The selected pups for hormone analysese were sacrificed by decapitation under isoflurane anesthesia in the pathology lab and blood was sampled for thyroid hormone analyses

On PND 21, all F2 generation pups were sacrificed under isoflurane anesthesia with CO2. After sacrifice, these pups were examined externally, eviscerated and their organs were assessed macroscopically.
The brain, spleen and thymus were weighed in one surplus F2 weanling per sex per litter (as a rule the first available male and female pup per litter). Pups’ relative organ weights were calculated from these weights and the weight of the living animal at sacrifice. Pups showing clinical symptoms or gross-morphological findings were examined using appropriate methods. Organs/tissues with gross-morphological findings were preserved in a suitable manner for potential histopathological examination. Pups that died or were sacrificed in a moribund state were eviscerated and examined for possible defects and/or the cause of death using appropriate methods. These animals were preserved for this purpose, if necessary.
All F1 and F2 pups which were not used for other purposes without any notable findings were discarded after their macroscopic evaluation.

Auditory startle response habituation in cohort 2A animals
On PND 24, the auditory startle response test was carried out in all animals of cohort 2A using the SR-LAB; STARTLE RESPONSE SYSTEM (San Diego Instruments, San Diego, CA, U.S.A.) in a randomized sequence. The examinations started in the morning. Age-appropriate sized enclosures were used. The animals were given a 5 minutes acclimation period in the response chamber with a 70 dBA background noise. Then the startle response was recorded
in 50 trials at a startle stimulus sound level of 120 dBA with a 5 - 10 second variable interval between the trials. Response was recorded for 50 milliseconds. Measurement was carried out with the light and ventilator switched on in the measurement chambers; no food or water was provided during the test. Data (maximum amplitude, latency to the peak of the response) were analyzed in 5 blocks of 10 trials each.

Functional observational battery (FOB) in cohort 2A animals
The FOB was carried out once, on PND 69, in all animals of cohort 2A. The examinations were generally started in the morning at about 10:00 h. The FOB was carried out in a randomized sequence. Before start of the FOB the animals were transferred separately into polycarbonate cage. Drinking water was provided ad libitum whereas no food was offered during the measurements. The FOB was started with passive observations without disturbing the rats, followed by removal from the home cage, open field observations in a standard arena and sensory motor tests as well as reflex tests. The findings were ranked according to their degree or severity, if applicable.

Home cage observation
The animals were observed for a short period (about 10-30 seconds) in their cages with the lids closed in the rack, while disturbing influences (touching of the cage and loud noises) were avoided. While other abnormalities were recorded, particular attention was paid to the parameters listed in table 2.


Open field observation
For observation, the animals were removed from their cages by the investigator and placed in a standard arena (50 × 50 × 25 cm). Besides noting other abnormalities, the parameters in table 2 were assessed.

Sensory-motoric test/Reflexes
The animals were removed from the open field and were subjected to the sensory motor and reflex tests listed in table 2.

Motor activity measurement (MA)
The measurement of motor activity (MA) was carried out on PND 69, in all animals of cohort 2A, on the same day when FOB was conducted. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. The animals were measured in individual clean polycarbonate cages with a small amount of bedding in randomized order (so that each session included males and females from different dose levels). Each cage was equipped with two sensor rings, the lower ring with 18 light beams and the upper ring (for counting of rearings) with 12 light beams. The number of beam interrupts and the rearing frequency were determined over 12 intervals, each lasting 5 minutes. On the respective testing days, the measurement sessions were always started at about 14:00 h, the
individual starting time was staggered by the time needed to place the animals in the cages. Test sessions were one hour long for each animal and began when the 1st beam was interrupted. No food or water was offered to the animals during these measurements. After the transfer of the last animal into the session, the measurement room was darkened.


ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:
Cohort 2A animals:
On postnatal day 77, all cohort 2A animals were weighed, subjected to deep anesthesia (pentobarbital) and sacrificed by perfusion fixation. The perfusion fixed animals were necropsied with regard to the question of neuropathology,
and the visible organs were assessed by gross pathology as accurately as is possible after a perfusion fixation. The cranial vault and the spinal cord were opened and the skin was removed from both hind extremities.

The following weights were determined (the brain was weighed after its removal but before further preparation):
1. Terminal body weight
2. Brain (including olfactory bulb)

The length and maximum width of the brain were measured in all animals. The length of the brain was measured from the cranial end of the cerebrum to the caudal end of the cerebellum; the width was taken at the level of the pituitary stalk.

Organs/tissue specimens in table 3 were carefully removed and processed histotechnically (some tissues only analyzed for high dose and control group, mid and low dose specimens preserved).

Morphometry analysis included thickness measurements of major brain layers (neocortex: frontal and parietal cortices, caudate nucleus/putamen, hippocampus, corpus callosum, cerebellum).
Measurements were carried out bilaterally in the left and right halves of the brain except for the corpus callosum and the cerebellum. For this purpose, homologous sections of three levels of the brain were prepared (3 serial
sections were prepared for each level and the most suitable section selected):
• Level 2, a coronal section, is used for the measurements of the neocortex: frontal (measurements 1 and 2) and parietal cortices (measurements 5 and 6), caudate nucleus/putamen (measurements 3 and 4), and corpus callosum (measurement 7)
• Level 3, a coronal section, is used for the measurements of the hippocampus
(measurements 8 and 9)
• Level 7, a longitudinal section, is used for the measurement of the cerebellum (8th lobus vermi cerebelli, measurement 10)
All 10 measurements were carried out in male and female animals of High dose and control groups.

Cohort 2B animals:
On postnatal day 22, cohort 2B animals were weighed, subjected to deep anesthesia (pentobarbital) and sacrificed by perfusion fixation. The perfusion fixed animals were necropsied with regard to the question of neuropathology,
and the visible organs were assessed by gross pathology as accurately as is possible after a perfusion fixation. The cranial vault and the spinal cord were opened, and the skin was removed from both hind extremities. In this state, the perfused animals were stored in neutrally buffered, 4% formaldehyde solution for at least 48 hours.

The following weights were determined (the brain was weighed after its removal but before further preparation):
1. Terminal body weight
2. Brain (including olfactory bulb)

Length and width of brain
The length and maximum width of the brain were measured in all animals. The length of the brain was measured from the cranial end of the cerebrum to the caudal end of the cerebellum; the width was taken at the level of the pituitary stalk.

Organs/tissue specimens in table 3 were carefully removed and processed histotechnically (some tissues only analyzed for high dose and control group, mid and low dose specimens preserved).

After completion of the neurohistopathological assessment by the study pathologist an internal fully-blinded peer review of the Cohort 2A (Developmental
Neurotoxicity) was performed. All slides were coded and re-examined by light microscopy without knowing the animal No. or the test group. The frequency and intensity of neuropathological changes were assessed. The presence of a
dose-response relationship was investigated in particular. The Peer Review included the thoracic cord of male and female animals in control and all treatment groups. Results presented in the pathology report reflect the consensus opinion of the study pathologist and the peer review pathologist.
Postmortem examinations (parental animals):
SACRIFICE
- All F0 generation parental animals and all F1 generation, rearing animals, cohort 1A were sacrificed. Before weaning of the F2 pups the F1 generation parental male animals were sacrificed. The F1 generation parental females were sacrificed, shortly after the F2 generation pups had been weaned. If animals were in a moribund state, they were sacrificed and necropsied.

-Pathological examinations of F0 generation parental animals and F1
generation, rearing animals, cohort 1A

All F0 generation parental animals and all F1 generation, rearing animals, cohort 1A were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology; special attention being given to the reproductive organs.

The tissues indicated in Table [4] were weighed. All paired organs were weighed together (left and right).
Tissues indicated in table 5 were prepared for microscopic examination. Some tissues were not analyzed in the low and mid dose groups.
The organs were trimmed according to the “Revised guides for organ sampling and trimming in rats and mice” (Ruehl-Fehlert et al., 2003; Kittel et al., 2004; Morawietz et al., 2004). For technical reasons, the left testis, left epididymis, and the eyes with optic nerves of all animals of the F0 parental control and high dose groups sacrificed at scheduled dates were embedded in paraplast after fixation. For the same reason, the ovaries of all F0 and cohort 1A females in all test groups were embedded in paraplast.
A correlation between gross lesions and histopathological findings was attempted. Special attention was given to stages of spermatogenesis in the male gonads. Special attention was also given to the synchrony of the morphology in ovaries, uterus, cervix, and vagina to the estrous cycle status. Reproductive organs of all F0 animals suspected of reduced fertility were subjected to
histopathological investigation. Whenever in the ovary the diagnosis: ”no abnormalities detected” was used, that implies that all different stages of functional bodies (especially corpora lutea) were present and normal.

After completion of the histopathological assessment by the study pathologist an internal peer review was performed including liver and thyroid glands of male and female animals in control and all treatment groups of F0 and F1A generation. Results presented in the pathology report reflect the consensus opinion
of the study pathologist and the peer review pathologist.

A differential ovarian follicle count (DOFC) was conducted in the control and high dose test group (cohort 1A females) according to Plowchalk et.al. (1993). In general, sections were prepared with 2 - 3 μm thickness and serial sections were taken every 100 μm to complete about 20 cut levels across the whole ovarian tissue. For the counting of primordial and growing follicles, H&E stained
slides were prepared from all cut levels. Counting was performed on slides digitalized with a Hamamatsu NanoZoomer 2.0 slide scanner using the Hamamatsu viewing software (NDP.view).


- Pathological examinations of F1 generation, rearing animals, cohort 1B
All cohort 1B animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology; special attention was given to the reproductive organs.

Organ weights indicated in table 4 were determined in all animals sacrificed on schedule. All paired organs were weighed together (left and right).

The uteri of all cohabited female F1 generation, rearing animals, cohort 1B were examined for the presence and number of implantation sites. The uteri of apparently nonpregnant animals or empty uterus horns were placed in 1% ammonium sulfide solutions for about 5 minutes in order to be able to identify early resorptions or implantations (SALEWSKI's method [Salewski,
1964]). Then the uteri were rinsed carefully in physiologic salt solution (0.9 % NaCl). When the examinations were completed, the uteri were transferred to the Pathology Laboratory for further processing.

Tissues indicated in table 5 were prepared for microscopic examination.
Postmortem examinations (offspring):
-Pathological examinations of surplus F1 generation pups on PND 22 (F1
weanlings not selected for cohorts)

All surplus F1 generation pups that were not used for the following organ weight
determinations were sacrificed under isoflurane anesthesia with CO2. The selected pups for organ weight determination were sacrificed by decapitation under isoflurane anesthesia. All animals were necropsied and assessed by gross pathology with special emphasis on the reproductive organs.

Organ weights
The following weights were determined in up to 10 animals per sex per group sacrificed on schedule:
1. Anesthetized animals (final body weight)
2. Brain
3. Spleen
4. Thymus (fixed)

Organ/Tissue fixation
The following organs or tissues of up to 10 animals per sex per group were fixed in 4% neutralbuffered formaldehyde solution:
1. All gross lesions
2. Brain
3. Mammary gland (male and female)
4. Spleen
5. Thymus
6. Thyroid glands

No histotechnical processing and examination was performed.

Post mortem analysis of neurotoxicological cohorts described above under litter observations.
Statistics:
Among others:

-Food consumption:
Simultaneous comparison of all dose groups with the control group using the
DUNNETT test (twosided) for the hypothesis of equal means

-Mating indices:
Pair-wise comparison of each dose group with the control group using
FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions

-Pup indices:
Pair-wise comparison of the dose group with the control group using
the WILCOXON test (one-sided-) with BONFERRONI-HOLM adjustment for the
hypothesis of equal medians.

-Blood parameters:
For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed
using WILCOXON-test (two-sided) for the hypothesis of equal medians For parameters with unidirectional changes: Pairwise comparison of each dose group
with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.

-Urinalysis parameters:
Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians. In case of exactly the same numbers of the dose group and the control, no statistical test is performed. And Non-parametric one-way analysis using
KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a
pairwise comparison of each dose group with the control group was performed
using WILCOXON-test (two-sided) for the hypothesis of equal medians.

-Spermanalysis parameters:
Pairwise comparison of each dose group with the control group using the
WILCOXON-test (one-sided) with Bonferroni-Holm adjustment for the
hypothesis of equal medians. For the percentage of abnormal sperms
(ABNORMAL5_C) values < 5 % were set to 5 % (cut off 5 %).
Reproductive indices:
Female mating index (%),
Female fertility index (%),
Gestation index (%),
Live birth index (%),
Postimplantation loss (%),
Offspring viability indices:
Viability index (%),
Lactation index (%)
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female F0 parental animals in any of the groups.
One high-dose male animal showed pilorection during study days 50 - 51. This
observation was considered not to be associated with the test compound.

There were no test substance-related clinical findings in any females of all dose groups during the gestation period for F1 litter. One low-dose female and one high-dose female had blood in bedding on GD 24 and 23, respectively. One sperm positive control female and one sperm positive low-dose female did not deliver F1 pups. One sperm positive low-dose female did not deliver F1 pups but had implants in the utero. These observations were considered not to be associated with the test compound.

There were no test substance-related clinical findings in all F0 females of all dose groups during the lactation period. One high-dose female had a pup palpable in abdomen after delivery on PND 0 and a complete litter loss on PND1. These observations were considered not to be associated with the test compound.
Mortality:
no mortality observed
Description (incidence):
There were no test substance-related or spontaneous mortalities in any of the groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights of all test substance treated F0 male animals were comparable to the concurrent control values throughout the entire study.

Mean body weights were statistically significantly below the concurrent control values for the high-dose F0 females during premating days 28 - 70 and during the entire gestation and lactation period (up to 9%, 14% and 14%, respectively).
The decrease in terminal body weight of females in the high dose group (1800 ppm) was regarded to be treatment-related.

Mean body weights of the low-and mid-dose F0 females were comparable to the concurrent control values throughout the entire study.


Body weight change of the high-dose F0 males was statistically significantly below the concurrent control values during study days 35 – 42, 49 - 63 and 112 - 119 (up to 33%), of the mid-dose males during study days 7 - 14, 35 - 42 and 49 - 56 (up to 29%) and for the low-dose males during study days 35 - 42 and 49 – 56 (up to 27%).

Body weight change of the high-dose F0 females was statistically significantly below the concurrent control values during premating days 0 - 7, 21 - 28, 42 - 49, 0 - 70 (up to 34%), during GD 0 - 14, 0 - 20 (up to 38%) and during PND 1 - 4 (about 72%), of the mid-dose females during premating days 42 - 49 (about 30%) and of the low-dose females during premating days 21 - 28 and GD 0 - 20 (about 22% and 10%, respectively).

A statistically significantly increased body weight change in the low-, mid- and high-dose males during study days 28 - 35 and 42 - 49, respectively, in the mid-dose males during study days 70 – 77 and in the mid- and high-dose females during PND 14 - 18 was considered as spontaneous in nature.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption of the high-dose F0 males was statistically significantly below the concurrent control values during study days 0 - 7 and 35 - 42 (up to 10%), of the mid-dose males during study days 35 - 42 and 49 - 56 (up to 9%) and for the low-dose males during study days 35 - 42 (about 9%). These changes were rather inconsistent and, thus, considered not to be treatment-related.

Food consumption of the high-dose F0 females was statistically significantly below the concurrent control values during the entire gestation and major parts (PND 1 - 18 and 1 - 21) of the lactation period (up to 14%, 16% and 13%, respectively).
Food consumption of the low- and mid-dose F0 females during the entire study and of the high dose females during the premating period was comparable to the concurrent control values.

A statistically significantly increased food consumption in the high-dose females during premating days 35 - 56 was considered to be spontaneous in nature.

The mean calculated test substance intakes were:
F0 males 200 ppm: 16.1 mg/kg bw/d;
F0 males 600 ppm: 48.6 mg/kg bw/d;
F0 males 1800 ppm: 146.1 mg/kg bw/d;

F0 females (premating) 200 ppm: 16.5 mg/kg bw/d;
F0 females (premating) 600 ppm: 49.8 mg/kg bw/d;
F0 females (premating) 1800 ppm: 182.9 mg/kg bw/d;

F0 females gestation period 200 ppm: 14.8 mg/kg bw/d;
F0 females gestation period 600 ppm: 44.7 mg/kg bw/d;
F0 females gestation period 1800 ppm: 133.7 mg/kg bw/d;

F0 females lactation period 200 ppm: 20.1 mg/kg bw/d;
F0 females lactation period 600 ppm: 58.1 mg/kg bw/d;
F0 females lactation period 1800 ppm: 174.7mg/kg bw/d;
Food efficiency:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period, in parental male rats of the high dose test group (1800 ppm) hemoglobin and hematocrit values were marginally but significantly decreased. Additionally, in males of this test group absolute reticulocyte counts were significantly increased whereas in females of this test group platelet counts were significantly decreased and prothrombin time (i.e.
Hepatoquick’s test, HQT) was significantly reduced. These alterations were regarded as treatment-related and adverse.

In addition, in parental males of the intermediate and high dose groups (600 and 1800 ppm) prothrombin time (HQT) was significantly increased. However, the change was not dose dependent and the values were within the historical control range (males, HQT 34.0-40.1 sec).

In females of the high dose group (1800 ppm) hemoglobin values, mean corpuscular volume (MCV) and mean corpuscular hemoglobin content (MCH) were significantly decreased, but hemoglobin and MCV values were within historical control ranges and MCH values were marginally below this
range (females, hemoglobin 8.6-10.0 mmol/L, MCV 50.7-55.1 fL, MCH 1.10-1.21 fmol). Only MCH as calculated red blood cell index was changed among these individuals, but not the measured red blood cell parameters (i.e. hemoglobin, hematocrit and red blood cell counts (RBC)).

In females of the low and intermediate dose groups (200 and 600 ppm) mean corpuscular hemoglobin concentration (MCHC) was significantly lower compared to controls and additionally in females of the low dose group prothrombin time (HQT) was significantly prolonged, but the alterations were not dose dependent. Therefore, these changes were regarded as incidental and not treatment related.

In high dose group females (1800 ppm) absolute monocyte counts were significantly increased and the mean was above the historical control range (females, absolute monocytes 0.05-0.11 Giga/L). This was the only changed differential blood cell parameter among these individuals and therefore, it was regarded as maybe treatment related but non-adverse.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period, in male and female rats of the intermediate and high dose groups (600 and 1800 ppm) alkaline phosphatase (ALP) activities and triglyceride values were significantly increased. Additionally, in rats of both sexes in the high dose group albumin levels were significantly
decreased. In males of this test group total protein values were also significantly decreased, whereas in females of the high dose group, globulin levels were significantly increased. In addition, in males of the high dose group alanine aminotransferase (ALT) activities were marginally, but significantly increased, and in females of this test group γ-glutamyl transferase (GGT) activities and cholesterol levels were significantly higher compared to controls. The mentioned
alterations were regarded as treatment related and adverse.


The following, significant changes were regarded as incidental and not treatment related, because the values were within historical control ranges:
In males: increased alanine aminotransferase (ALT) activities at 600 ppm, increased alkaline phosphatase (ALP) activities at 200 ppm, decreased total bilirubin values at 200 and 1800 ppm;

In females: in the low and intermediate dose groups decreased albumin values
(males, ALT 0.64-0.86 μkat/L, ALP 0.96-1.57 μkat/L, total bilirubin 1.08-1.80 μmol/L; females, albumin 35.28-40.13 g/L). In females at 200 and 600 ppm creatinine values were significantly increased and in females of the intermediate test group calcium values were significantly lower compared to controls, but both alterations were not dose dependent. Therefore, these mentioned alterations were regarded as incidental and not treatment related.

In females animals at 200 ppm alkaline phosphatase (ALP) activities and in males of this test group triglyceride values were already significantly increased. Both parameter values were above historical control ranges (females ALP, 0.57-1.10 μkat/L; males, triglycerides 0.59-0.97 mmol/L). However, in this test group these were the only relevantly changed parameters and therefore, the changes were regarded as treatment related but non adverse.


In F0 males of test groups of the low, intermediate, and high dose test groups (200, 600 and 1800 ppm) T4 values were significantly decreased, although not dose dependent.
TSH values were not statistically significantly changed. The T4 values of the low, intermediate and high dose test groups were below the historical control range,
TSH values of all three groups above this range. However, control T4 values were also at the lower border of the historical range and TSH control group values at the upper border of this range (males, T4 44.65-73.22 nmol/L, TSH 4.32-10.04 μg/L).
Therefore, in combination with histopathological findings in the thyroids in the low, intermediate, and high dose groups, T4 decreases and TSH increases in all three test groups were regarded as treatment related and adverse.


In F0 females the low, intermediate, and high dose groups (200, 600 and 1800 ppm) TSH values were significantly increased. T4 values were not changed. TSH values of females in the low and high dose groups were above the historical control range, those of the intermediate dose groups were within this range. T4
values of all test groups, including the control group were below the historical control range (females, T4 28.48-43.26 nmol/L, TSH 3.23-6.94 μg/L). Therefore, at least TSH increases in test groups the low, intermediate and high dose groups in combination with histopathological findings in the thyroids are regarded as treatment-related and adverse. T4 values were not changed within the study, and
lower levels as historical controls in all samples including controls may be accidentally.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among urinalysis parameters were observed.
In females of the intermediate test group (600 ppm) urine pH values were significantly increased, but this change was not dose dependent. Therefore, this alteration was regarded as incidental and not treatment related.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Male animals of the intermediate and high dose groups (600 and 1800 ppm) revealed a minimal to moderate centrilobular liver cell hypertrophy. In addition, most of these animals showed a microvesicular vacuolation in the transition region between centrilobular and midzonal area. Females of all test groups showed a centrilobular liver cell hypertrophy. Additionally, six female animals of the high dose group revealed a diffuse hepatocellular hypertrophy. These findings were regarded to be treatment-related.

The livers of two animals which were stained with Oil Red O (ORO) to detect neutral lipids, did not show a positive reaction.
The livers of two animals which showed a microvesicular vacuolation in the high dose group showed a positive reaction when stained with ORO. Therefore, the
microvesicular vacuolation was shown to be neutral lipids (fatty change).

Males and females of all test groups revealed a higher incidence of thyroid follicular cell hypertrophy/hyperplasia. Animals of the low dose group showed only minimally increased incidences compared to control. In addition, there was an increase of more floccular, basophilic colloid (altered colloid) in males of the high dose group (1800 ppm) and females of all test groups. These findings were considered as treatment-related.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

The two female animals, which were not pregnant did not show relevant
histopathological findings consistent with impaired fertility. The male mating partner showed diffuse tubular degeneration in the testicle and aspermia in the epididymis. The second male mating partner revealed multifocal tubular degeneration in the testicle and oligospermia in the epididymis. The findings in the testes of the two male animals were regarded to be the cause of having no offspring. This was regarded as incidental.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrous cycle data, generated during the last 3 weeks prior to mating to produce the F1 litter, revealed regular cycles in the females of all test groups including the control. The mean estrous cycle duration was similar: 4.1 / 4.4 / 4.0 and 4.1 days in control, low, intermediate, and high dose groups
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Concerning motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as sperm head counts in the testis and in the cauda epididymidis of parental males no treatment-related effects were observed.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100% in all test groups.

Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter.

One control male and one low-dose male did not generate F1 pups.
Thus, the male fertility index ranged between 96% and 100%, reflecting the normal range of biological variation inherent in the strain of rats used for this study.
The affected Control male showed diffuse tubular degeneration in the testicle and aspermia in the epididymis. The Low-dose male revealed multifocal tubular degeneration in the testicle and oligospermia in the epididymis. The findings in the testes of these two male animals were regarded to be the cause of not producing offspring. Both were regarded as incidental.

The female mating index calculated after the mating period for F1 litter was 100% in all test groups.

The mean duration until sperm was detected (GD 0) varied between 2 and 3 days without any relation to the dose.

All female rats delivered pups or had implants in utero with the following exception:
One control group animals did not become pregnant.
One low dose group female did not become pregnant.
The apparently infertile female rats did not show histopathological findings that could explain infertility. Both non-pregnancies were caused by the male mating partners.

The fertility index ranged between 96% and 100% without any relation to the dose.


The mean duration of gestation was 22.2, 22.1, 21.7** and 22.2 days in the control, low, intermediate and high dose groups, respectively. Although the marginally lower value in the mid-dose group is slightly outside the historical control range (HCD = 21.8 - 22.3), no biological significance is ascribed to it, as there is no dose response and delivery dates of GD 21-23 are normal in the
present rat strain.

The gestation index was 100% in control and the intermediate and high dose groups and 95.8% in the low dose group.

The mean number of implantation sites was statistically significantly below the concurrent control values in the high-dose group (12.1 / 11.0 / 11.9 and 10.7** implants/dam in control, low, intermediate, and high dose groups, respectively) and slightly below the historical control range (HCD = 11.1 - 15.3
implants/dam).

There were no indications for test substance-induced intrauterine embryo-/fetolethality since the postimplantation loss did not show any statistically significant differences between the groups (4.8% / 9.0% / 3.4% and 10.2% in control, low, intermediate, and high dose groups, respectively),

As a consequence of the lower number of implants the mean number of F1 pups delivered per dam (average litter size) was statistically significantly below the concurrent control values in the high-dose group (12.0 / 11.3 / 11.5 and 9.6** pups/dam, in control, low, intermediate, and high dose groups, respectively), and outside the historical control range (HCD = 10.3 - 14.9 pups/dam).


The rate of liveborn pups indicated by live birth indices was 97.6% / 99.6% / 100% and 98.7% in control, low, intermediate, and high dose groups, respectively, showing no significant differences between the groups.

The number of stillborn pups was not significantly different between the test groups.
Dose descriptor:
NOAEL
Remarks:
600 ppm
Effect level:
54 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
other: NOAEL for reproductive performance in F0 generation
Dose descriptor:
LOAEL
Remarks:
200 ppm
Effect level:
18 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Remarks on result:
other: NOAEL for general systemic toxicity in the F0 parental rat
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female F1B parental animals in any of the groups.

One mid-dose male animal showed a skin lesion (right shoulder region) during study days 35 - 55. This observation was considered not to be associated with the test compound.

There were no test substance-related clinical findings in any females of all dose groups during the gestation period for F2 litter.

One high-dose female showed piloerection during GD 16 - 17.
Two sperm positive control females, one sperm positive low-dose female and one sperm positive mid-dose female did not deliver F1 pups.

These observations were considered not to be associated with the test compound.

There were no test substance-related clinical findings in all F1B females of all dose groups during the lactation period.
Mortality:
no mortality observed
Description (incidence):
There were no test substance-related or spontaneous mortalities in any of the groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights of the high-dose male rats were statistically significantly below the concurrent control values on study day 7 and during study days 35 - 104 (up to 7%).

Mean body weights were statistically significantly below the concurrent control values for the high-dose F1B females during premating days 7 - 70 and during the entire gestation and lactation period (up to 9%, 14% and 17%, respectively).

Mean body weights of the low-and mid-dose F1B males and females were comparable to the concurrent control values throughout the entire study.

Body weight change of the high-dose F1B males was statistically significantly below the concurrent control values during study days 0 - 7, 28 - 35, 42 - 49 and 63 - 70 (up to 53%) and of the mid-dose males during study days 77 - 84 (about 45%).

Body weight change of the high-dose F1B females was statistically significantly below the concurrent control values during premating days 0 - 7, 21 - 28, 49 - 56, 63 - 70, 0 - 70 (up to 55%), during GD 0 - 14, 0 - 20 (up to 41%) and during PND 1 - 4 (about 54%) and for the middose females during PND 1 - 4 (about 30%).

The decrease in terminal body weight in males and females of the high dose group (1800 ppm) was regarded to be treatment-related.

Body weight change of the low-dose males and females were comparable to the concurrent control values throughout the entire study.

The statistically significantly increased body weight change in the mid-dose males during study days 14 - 21 and 91 - 98 and in the high-dose females during PND 14 - 18 was considered as spontaneous in nature.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption of all test substance treated male rats was comparable to the concurrent control values throughout the entire study.

Food consumption of the high-dose F1B females was statistically significantly below the concurrent control values during premating days 0 - 7, 42 - 49, 56 - 70 and 0 - 70 (up to 10%) and during the entire gestation and lactation periods (up to 16% and 22%, respectively).

Food consumption of the low- and mid-dose F1B females was comparable to the concurrent control values during the entire study.

The mean calculated test substance intakes were:
F0 males 200 ppm: 17.0 mg/kg bw/d;
F0 males 600 ppm: 51.7 mg/kg bw/d;
F0 males 1800 ppm: 159.8 mg/kg bw/d;

F0 females (premating) 200 ppm: 18.0 mg/kg bw/d;
F0 females (premating) 600 ppm: 53.6 mg/kg bw/d;
F0 females (premating) 1800 ppm: 164.3 mg/kg bw/d;

F0 females gestation period 200 ppm: 15.8 mg/kg bw/d;
F0 females gestation period 600 ppm: 47.1 mg/kg bw/d;
F0 females gestation period 1800 ppm: 139.1 mg/kg bw/d;

F0 females lactation period 200 ppm: 20.2 mg/kg bw/d;
F0 females lactation period 600 ppm: 59.2 mg/kg bw/d;
F0 females lactation period 1800 ppm: 177.0 mg/kg bw/d;
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Also, the increase of absolute liver weight in females and relative liver weight in males and females of the high dose group (1800 ppm) was thought to be
treatment-related.

The increase of absolute uterus weight of low dose group females (200
ppm) was considered to be incidental. All other reduced absolute organ weights in the high dose group (1800 ppm) were regarded to be a consequence to the reduced terminal body weight and no direct effect of the test substance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.



The two female animals which were not pregnant, as well as their male mating partners did not show relevant gross lesions. One male mating partner
revealed size reduction of testes and epididymides. In histology this male animal
revealed a moderate multifocal degeneration of the testes and oligospermia in the
epididymides.
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
Estrous cycle data, generated during 2 weeks, revealed regular cycles in the females of all test groups including the control. The mean estrous cycle duration was comparable between the groups: 3.9 / 3.9 / 4.0 and 4.0 days in the control, low, intermediate and high dose groups, respectively.

Estrous cycle data, generated during the last 3 weeks prior to mating to produce the F2 litter, revealed regular cycles in the females of test groups 10 - 13. The mean estrous cycle duration was: 4.0 / 4.0 / 4.0 and 4.3** (**:p<=0.01) days in the control, low, intermediate, and high dose groups, respectively.

The slightly prolonged average in test group 13 is due to a 5-day cycle in 5 females, which is still normal in the tested rat strain. The average cycle length is within the historical control range (HCD = 3.9 - 4.6) and a comparable increase was not observed in the corresponding cohort 1A females.
Thus, the apparent prolongation is considered as a spurious finding.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Concerning motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as sperm head counts in the testis and in the cauda epididymidis of F1A males no treatment-related effects were observed.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
For all F1B parental males, which were placed with females to generate F2 pups, copulation was confirmed. Thus, the male mating index was 100% in all test groups.

Fertility was proven for most of the F1B parental males within the scheduled mating interval for F2 litter.
Two control males, one low-dose male and one mid-dose male did not generate F2 pups. Thus, the male fertility index ranged between 92% and 100% without showing any relation to the dose. These data reflect a normal range of biological variation in the strain of rats used for this study.

The apparently infertile male rats did not show relevant gross lesions. One control male revealed size reduction of testes and epididymides. In histopathology this male animal revealed a moderate multifocal degeneration of the testes and oligospermia in the epididymides.



The female mating index calculated after the mating period for F2 litter was 100% in all test groups.

The mean duration until sperm was detected (GD 0) varied between 2.3 and 3.4 days without any relation to the dose.

All female rats delivered pups or had implants in utero with the following exception:
2 control group females did not become pregnant. One low dose group female did not become pregnant. One female of the intermediate dose group did not become pregnant.

The apparently infertile female rats did not show histopathological findings that could explain infertility.

The fertility index ranged between 92% and 100% without any relation to the dose.

The mean duration of gestation values varied between 21.7 and 22.0 days without any relation to the dose.

The gestation index was 100% in all test groups.

The mean number of implantation sites was statistically significantly below the concurrent control values in the high-dose group (12.3 / 11.8 / 11.2 and 10.2** (**:p<=0.01) implants/dam in the control, low, intermediate, and high dose groups, respectively) and slightly below the historical control range (HCD = 11.1
- 15.3 implants/dam).
It was just inside the historical control range in the mid-dose group.

There were no indications for test substance-induced intrauterine embryo-/fetolethality since the postimplantation loss did not show any statistically significant differences between the groups (2.0% / 4.5% / 3.3% and 4.0% the control, low, intermediate, and high dose groups, respectively).

The mean number of F2 pups delivered per dam (average litter size) was statistically significantly below the concurrent control values in the mid- and high-dose group (12.0 / 11.2 / 10.8* (*:p<=0.05) and 9.8** (**:p<=0.01) pups/dam, respectively in test groups 10 - 13). The lower average litter size is for both groups considered to be a consequence of a lower number of implants. While the litter size of the high-dose group was outside the historical control range
(HCD = 10.3 - 14.9 pups/dam), it is still within range in the mid-dose group.

The rate of liveborn pups indicated by live birth indices was 99.6% / 99.6% / 99.6% and 98.0%the control, low, intermediate, and high dose groups, respectively.

The number of stillborn pups was not significantly different between the test groups.
Dose descriptor:
NOAEL
Remarks:
600 ppm
Effect level:
54 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
other: NOAEL for reproductive performance in F1 generation
Dose descriptor:
NOAEL
Remarks:
200 ppm
Effect level:
18 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
haematology
clinical biochemistry
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Remarks on result:
other: General, systemic toxicity in the F1 rats
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related adverse clinical signs were observed in any of the F1 generation pups of the different test groups.
One low-dose male pup had a small eye during PND 21 – 22 and one
low-dose female pup had an absent eye from PND 20 onwards. These
observations were considered not to be associated with the test compound.

No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female animals in any of the F1 rearing animal test groups.
One control male animal showed a skin lesion (left shoulder region) during study
days 36 - 49 and one high-dose female animal had an absent vaginal opening from study day 34 onwards till the end of the study. These observations were considered not to be associated with the test compound.

One control male animal showed scratching and twitching (grade: moderate) during study days 28 - 49, respectively.
Mortality / viability:
no mortality observed
Description (incidence and severity):
The viability index indicating pup survival during early lactation (PND 0 - 4) varied between 98.7% / 98.0% / 98.5% and 93.3% in the control, low, intermediate, and high dose groups without showing significant differences between the groups.

The number of cannibalized and dead F1 pups were evenly distributed about the groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study.

The lactation index indicating pup survival on PND 4 - 21 was 99.3% / 99.6% / 100% and 100% in the control, low, intermediate, and high dose groups without showing any association to the treatment.

No mortality was observed in F1 rearing animals.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights of the high-dose pups of both sexes were statistically significantly and consistently below the concurrent control beginning at PND 7 until PND 21 (ranging between 10% and 13% below control).

Mean body weight change of high-dose male and female pups and both sexes combined was statistically significantly below the concurrent control values
during PND 4 - 21 and 1 - 21 (up to 22%, 19% and 20%, respectively).

Mean body weights of the mid-dose pups of both sexes were below the concurrent control on PND 4, 7 and 21 (ranging between 6% and 8% below control).

The mean body weight change of mid-dose female pups and both sexes combined was statistically significantly below the concurrent control values during PND 1 - 4 and 14 - 21 (up to 10%, respectively).


No test compound-related influence on F1 pup body weights/pup body weight change were noted in all pups of the low-dose group.


Mean body weights of F1 rearing animals of the high-dose male and female rats were statistically significantly below the concurrent control values during the entire study (up to 10% and 7%, respectively).

Mean body weights of the F1 rearing low-and mid-dose males and females were comparable to the concurrent control values throughout the entire study.

Body weight change of the high-dose F1A males was statistically significantly below the concurrent control values during study days 7 - 14, 21 - 28, 35 - 42 and 0 - 56 (up to 27%), of the mid-dose males during study days 28 - 35 (about 30%) and for the low-dose males during study days 7 - 14 and 35 - 42 (up to 39%).

Body weight change of all test substance-treated F1 rearing female rats was comparable to the concurrent control values throughout the entire study.
The statistically significantly increased body weight change in the low- and mid-dose males during study days 14 - 21, respectively, and in the low- and high-dose males during study days 28 - 35, respectively, were considered as spontaneous in nature.

The terminal (final) body weight decrease in high dose group males and females (1800 ppm) was regarded to be treatment-related.

In F1 cohort 2A animals, mean body weights of the high-dose male rats were statistically significantly below the concurrent control values on study day 21 (about 10%).

Mean body weights and body weight change of F1 cohort 2A animals of the low-and mid-dose males and all test substance-treated females were comparable to the concurrent control values throughout the entire study.

The statistically significantly increased body weight change in the low-dose males and mid-dose females during study days 21 – 28, respectively, was considered as spontaneous in nature.

Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
In F1 rearing animals, food consumption of the high-dose males was statistically significantly below the concurrent control values during study days 7 - 14 and 21 - 28 (up to 11%) and of the mid-dose males during study days 28 - 35 (about 7%). These changes were rather inconsistent and, therefore, considered not to be treatment-related.

Food consumption of low-dose male rats and all test substance-treated female rats (200, 600 and 1800 ppm) was comparable to the concurrent control values throughout the entire study.

Food consumption of F1 rearing animals:
Males 200 ppm: 18.5 mg/kg bw/d;
Males 800 ppm: 56.5 mg/kg bw/d;
Males 1600 ppm: 174.8 mg/kg bw/d;

Females 200 ppm: 18.7 mg/kg bw/d;
Females 800 ppm: 56.6 mg/kg bw/d;
Females 1600 ppm: 169.7 mg/kg bw/d;
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At PND 90 in rats of both sexes of the high dose test group (1800 ppm) hemoglobin and hematocrit values were significantly decreased. Additionally, in females of this test group mean corpuscular volume (MCV) and mean corpuscular hemoglobin content (MCH) were significantly decreased and prothrombin time (i.e., Hepatoquick’s test, HQT) was significantly shortened. In males of the high dose test group, absolute reticulocyte counts were significantly increased, whereas platelet counts were significantly decreased. These alterations were regarded as treatment related and adverse.

In females of the intermediate test group (600 ppm) MCH was already significantly decreased. The mean was below the historical control range (females, MCH 1.09-1-1.15 fmol). However, no measured red blood cell parameter (i.e. red blood cell (RBC) counts, hemoglobin and hematocrit values) was changed among these individuals. Therefore, this alteration was regarded as incidental and not treatment related.

The following significant changes were regarded as incidental and not treatment related, because the values were within historical control ranges:
Females of the low, intermediate, and high dose test groups (200, 600 and 1800 ppm) showed decreased platelet counts;
Females of the high dose test group had increased relative monocyte and large unstained cell (LUC) counts and decreased relative basophil counts;
Females of the low dose test group showed decreased relative basophil counts (females, platelets 653-859 Giga/L, relative monocyte 1.4-2.4 %, relative LUC 0.2-0.6 %, relative basophils 0.2-0.4 %);
In females of the intermediate test group red blood cell (RBC) counts were significantly increased and in males of the low and intermediate test groups prothrombin time was significantly prolonged, but the changes were
not dose dependent. Therefore, these alterations discussed in this paragraph were regarded as incidental and not treatment related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At PND 90 in rats of both sexes of the intermediate and high dose test groups (600 and 1800 ppm) alkaline phosphatase (ALP) activities were significantly increased whereas albumin values were significantly decreased. Additionally, in males of these test groups total protein and globulin values were significantly decreased. In high dose group male and female rats total bilirubin values were significantly decreased. Additionally, in females of this test group γ-glutamyl transferase (GGT) activities, triglyceride, cholesterol and globulin values were significantly
increased. In females of the low dose group (200 ppm) ALP activities were already significantly increased, and albumin values were decreased. These alterations were regarded as treatment related and adverse.


The following significant changes were regarded as incidental and not treatment related, because the values were within historical control ranges:
Females of the high dose group (1800 ppm) had decreased sodium and chloride values (females, sodium 141.7-144.0 mmol/L, chloride 98.8-102.9 mmol/L).
In males of the intermediate test group (600 ppm) alanine aminotransferase (ALT) activities were significantly increased and in males of the low and intermediate test groups (200 and 600 ppm) inorganic phosphate values were significantly increased, but both alterations were not dose dependent.
Therefore, the changes discussed in this paragraph were regarded as incidental and not treatment related.



T4 and TSH values in PND4 male and female pups were not statistically significantly changed. However, in the high dose group (1800 ppm) hormone values of only 2 pups of each sex could be measured.
T4 values in males of all test groups including the control group were below the
historical control range whereas in female PND4 pups T4 values of the control group and the low dose group were at the low border of this range and T4 values in female PND4 pups of the intermediate and high dose groups were below this range. TSH values of males and females in all test groups were within the historical control range (PND4, males T4 18.36-36.79 nmol/L, TSH 3.19-5.25 μg/L;
females T4 17.88-34.51 nmol/L, TSH 3.05-6.36 μg/L).
TSH values in male and female PND22 pups of the intermediate and high dose groups (600 and 1800 ppm) were significantly increased. However, T4 and TSH values of all test groups including the controls were within historical control ranges (PND22, males T4 50.57-71.39 nmol/L, TSH 3.40-4.87 μg/L; females T4 44.85-73.70 nmol/L, TSH 2.92-5.13 μg/L). Therefore, the TSH level increased in PND pups the intermediate and high dose group were regarded as incidental and not treatment related.

T4 values in F1A males of the intermediate and high dose groups (600 and 1800 ppm) were significantly decreased, whereas TSH values of high dose group males were significantly increased. T4 values of high dose group F1A males were below the historical control range, whereas TSH values in this test group were within the upper end of its historical control range.
T4 values in intermediate dose group males were within the historical control range (F1 males, T4 49.46-88.73 nmol/L, TSH 2.61-9.90 μg/L). Therefore, in high dose group F1A males (1800 ppm) T4 decreases and TSH increases were regarded as treatment related and adverse, which correlates well with the histopathological findings in the thyroid of this group.

In intermediate and high dose group F1A females (600 and 1800 ppm) TSH values were increased (in the intermediate dose group not statistically significantly) and also at the upper border (intermediate dose group) or above
the historical control range (high dose group) (F1 females TSH 2.82-6.51 μg/L).

T4 values were only significantly decreased in intermediate dose group F1A females (600 ppm). In contrast, in females of the high dose group, T4 values were higher compared to study controls. T4 values of the intermediate dose group were below the historical control range (F1 females, T4 34.34-65.01 nmol/L). Therefore, in F1A females of the intermediate and high dose groups (600 and 1800 ppm) TSH increases (means 36 % and 59% increases compared to study controls) as well as T4 decrease in the intermediate dose group in combination
with histopathological effects in the thyroids were regarded as treatment-related and adverse.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related, adverse changes among urinalysis parameters were observed. In F1A males of the high dose group urine pH values were significantly decreased.

However, without any other change of the urine parameters this change was regarded as maybe treatment related, but non-adverse.
Sexual maturation:
effects observed, non-treatment-related
Description (incidence and severity):
Each female F1 pup, which was selected to become a rearing female, was evaluated for commencement of sexual maturity.
The first day when vaginal opening was observed was PND 28, the last was PND 41.
The mean number of days to reach the criterion in the control and
200/100, 600/300 and 1800/900 ppm test groups was 31.0; 31.1; 31.3 and 31.8* (* = p≤0.05) days, respectively.
The mean body weight on the day, when vaginal opening was recorded,
amounted to 96.0 g, 97.1 g, 94.3 g and 92.2 g in the control, low, intermediate, and high dose groups.
The apparent delay in the high dose group is small, the timing well within the historical control range (HCD: 29.5 - 38.8 days) and related to lower body weights of the affected offspring. Thus, the observed statistical change is considered not toxicologically relevant.

Each male F1 pup, which was selected to become a rearing male, was evaluated for commencement of sexual maturity. The first day when preputial separation was observed was PND 38, the last was PND 48. The mean number of days to reach the criterion in the control and 200/100, 600/300 and 1800/900 ppm test groups was 42.1, 41.5, 42.3 and 43.5** (** = p≤0.01) days, respectively.
The mean body weight on the day, when preputial separation was recorded, amounted to 181.2 g, 176.7 g, 180.9 g and 175.3 g in the control, low, intermediate, and high dose groups.

The apparent delay in the high dose group is small, the timing well within the historical control range (HCD: 40.1 - 45.2 days) and related to lower body weights of the affected offspring. Thus, the observed statistical change is considered not toxicologically relevant.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
The anogenital distance and anogenital index of all test substance treated male and female pups was comparable to the concurrent control values.
Nipple retention in male pups:
effects observed, non-treatment-related
Description (incidence and severity):
The percentage of male pups having nipples/areolae was not influenced by the test substance when examined on PND 13.
During the re-examination on PND 20 two high-dose male pups of one litter exhibited two nipple/areola anlagen, respectively. This is considered to be a
spontaneous event.
No nipples/areolae were detected in any other male pup of all test groups.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
A liver weight increase of females of the intermediate test group (relative) and high dose group (absolute and relative) (600 and 1800 ppm) and the increase of absolute and relative thyroid gland weight in intermediate and high dose group females of (600 and 1800 ppm) were considered to be treatment-related.

The decrease of absolute adrenal gland weight in high dose group females (1800 ppm) was slightly beneath historical control values, but it was still regarded to be
secondary to the body weight decrease. Especially, as the relative organ weight was not significantly changed, and no histopathologic finding was observed that could explain the weight decrease. All other decreased absolute and increased relative organ weights were considered secondary to the decrease in terminal body weight and not to be a direct effect of the test substance.

Regarding data from surplus F1 generation pups on PND 22, the decrease of terminal body weight in high dose group males and females (1800 ppm) was
regarded to be treatment-related. The increase of terminal body weight in low dose group males (200 ppm) did not show a dose response relationship and was therefore considered to be incidental. The decrease of absolute thymus weight in high dose group males and the increase in relative brain weight in high dose group males (1800 ppm) were regarded to be a consequence to the reduced terminal body weight and no direct effect of the test substance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A few F1 pups showed spontaneous findings at gross necropsy, such as absent testis, large spleen, post mortem autolysis and empty stomach. These findings occurred without any relation to dosing. Thus, all these findings were considered not to be associated with the test substance.

All findings in F1A animals occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
F1A cohort males of all test groups revealed a minimal to moderate centrilobular liver cell hypertrophy. In addition, some male animals of the intermediate and high dose groups (600 and 1800 ppm) showed a microvesicular vacuolation (fatty change) also in the centrilobular region, but at the rim towards the midzonal area. Also, intermediate and high dose female animals (600 and 1800 ppm) showed
centrilobular liver cell hypertrophy. These findings were regarded to be treatment-related.

High dose group males (1800 ppm) and intermediate and high dose group females (600 and 1800 ppm) revealed a higher incidence of thyroid follicular cell hypertrophy/hyperplasia. In addition, there was an increase of floccular, basophilic colloid (altered colloid) in high dose group males and females (1800 ppm). These findings were considered as treatment-related.

In the low and intermediate dose groups (200 and 600 ppm) of males the minimally increased incidences of hypertrophy/hyperplasia of follicular cells compared to control were regarded to be incidental, as no dose- response relationship was present.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.


The results of the differential ovarian follicle count (DOFC) – comprising the numbers of primordial and growing follicles, as well as the combined incidence of primordial plus growing follicles – did not reveal significant differences between the control group and animals of the high dose group.


Neurohistopathological examination of F1 pups (Cohort 2A animals)

The mean absolute and relative organ weight parameters did not show significant differences when* compared to the control group.

The length of the brain of high dose group males was significantly decreased. Width measurements were without any findings. The decreased length of the brain was regarded to be a secondary effect to the decreased terminal body weight/size of the animals rather than a direct effect on the brain itself as the relative brain weight was comparable to control animals (+2%).

Treatment-related findings were observed in the thoracic cord of male animals of the high dose group. In the thoracic cord of high dose group males, there was an increased incidence of focal to multifocal axonal degeneration in the white matter. This was characterized by digestion chambers with occasional pyknotic nuclei and presence of gitter cells (macrophages with foamy cytoplasm interpreted as ingested myelin debris).

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.



Neurohistopathological examination of F1 pups (Cohort 2B animals)

When compared to the control group, the mean relative weight of the brain was
significantly increased in males of the intermediate and high dose test groups and females of the high dose group. The decreased terminal body weights of males of the intermediate and high dose test groups and females of the high dose group were regarded to be treatment-related. The increased relative brain weights in these groups in males and females was assessed as a secondary effect to the body weight decrease.

All length and width measurements were without any findings.

No treatment-related histopathological findings were seen. All lesions are regarded as incidental and/or spontaneous.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Pup number and status at delivery

As a consequence of the lower number of implants the mean number of F1 pups delivered per dam (average litter size) was statistically significantly below the concurrent control values in the high-dose group (12.0 / 11.3 / 11.5 and 9.6** pups/dam, respectively in the control, low, intermediate and high dose groups), and outside the historical control range (HCD = 10.3 - 14.9 pups/dam).

The rate of liveborn pups indicated by live birth indices was 97.6% / 99.6% / 100% and 98.7% in the control, low, intermediate and high dose groups, respectively.

The mean number of liveborn pups was statistically significantly below the concurrent control values in the high-dose group (11.7 / 11.3 / 11.5 and
9.4** pups/dam, respectively in the control, low, intermediate and high dose groups), as a consequence of the lower number of implants in this group.

The number of stillborn pups was not significantly different between the test groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study.

The sex distribution and sex ratios of live F1 pups on the day of birth and on PND 21 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No influence of the test substance on auditory startle habituation (maximum amplitude and latency) was observed in any male or female animal in all treated groups.

No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation.

The open field observations did not reveal any test substance-related findings in male and female animals of all test groups.

There were no test substance-related findings in male and female animals of all test groups.

No statistically significant changes on motor activity data (summation of all intervals) was observed in the male and female animals of all dose groups in comparison to the concurrent control group.
Dose descriptor:
NOAEL
Remarks:
600 ppm
Generation:
F1 (cohort 2A)
Effect level:
54 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: NOAEL for developmental neurotoxicity
Dose descriptor:
NOAEL
Remarks:
200 ppm
Generation:
F1
Effect level:
18 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: NOAEL for developmental toxicity
Clinical signs:
no effects observed
Description (incidence and severity):
There were no test substance-related adverse clinical signs observed in any of the F2 generation pups of the different test groups.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The viability index indicating pup survival during early lactation (PND 0 - 4) was statistically significantly below the concurrent control values in the mid-dose group (100% / 100% / 97.0%* / 98.3% in the control, low, intermediate, and high dose groups, respectively).
As there is no dose response the marginally lower mid-dose value is considered to be an incidental finding. The number of cannibalized and dead F2 pups were evenly distributed about the groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study.

The lactation index indicating pup survival on PND 4 - 21 was 100% in all test groups.

The lactation index indicating pup survival on PND 4 - 21 was 99.3% / 99.6% / 100% and 100% in the control, low, intermediate, and high dose groups, respectively without showing any association to the treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights of the high-dose pups of both sexes combined were statistically significantly and consistently below the concurrent control beginning at PND 7 until PND 21 (ranging between 12% and 17% below control).

Mean body weight change of high-dose pups combined was statistically significantly below the concurrent control values during PND 4 - 21 and 1 - 21 (up to 22% below control).

No test compound-related influence on F2 pup body weights/pup or body weight change were noted in all pups of the low- and mid-dose group.

A statistically significantly increased body weight change in the low-dose pups during PND 1 - 4 was considered as spontaneous in nature.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
The anogenital distance and anogenital index of all test substance treated male and female pups was comparable to the concurrent control values.
Nipple retention in male pups:
effects observed, non-treatment-related
Description (incidence and severity):
The percentage of male pups having nipples/areolae was not influenced by the test substance when examined on PND 13.

Although there was no difference in the percentage of male pups having nipples/areolae the mean nipple number was statistically significantly above the concurrent control values in the high-dose male pups when examined on PND 13 (2.6 / 1.8 / 2.9 and 4.1* (*:p<=0.05) in the control, low, intermediate, and high dose groups, respectively).
This is considered to be the consequence of a general delay of pup development, rather than a specific effect on hormonal homeostasis.

During the re-examination on PND 20 no nipples/areolae were detected in any male pup of all test groups.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Reduced absolut organ weights were observed in spleen and thymus weights in male and female animals. Relative organ weights of brain and spleen in male and female animals.

All statistically significant absolute and relative weight changes in the high-dose group are related to the reduced body weight of the corresponding pups at weaning.
Occasional weight changes in the low-dose group are incidental findings.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A few F2 pups showed spontaneous findings at gross necropsy, such as discolored testis, small testis, dilated renal pelvis, absent epididymis, diaphragmatic hernia and partly cannibalized. These findings occurred without any relation to dosing. Thus, all these findings were considered not to be associated to the test substance.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Pup number and status at delivery
As a consequence of the lower number of implants the mean number of F2 pups delivered per dam (average litter size) was statistically significantly below the concurrent control values in the mid- and high-dose group (12.0 / 11.2 / 10.8* (*:p<=0.05) and 9.8** (**:p<=0.01) pups/dam, respectively in the control, low, intermediate, and high dose groups). While the litter size of the high-dose group was outside the historical control range (HCD = 10.3 - 14.9 pups/dam), it is still within range in the mid-dose group.

The rate of liveborn pups indicated by live birth indices was 99.6% / 99.6% / 99.6% and 98.0% in the control, low, intermediate, and high dose groups, respectively.

The mean number of liveborn pups was statistically significantly below the concurrent control value in the high-dose group (12.0 / 11.2 / 10.8 and
9.6** pups/dam, respectively in the control, low, intermediate, and high dose groups), as a consequence of the lower number of implants in this group.
The number of stillborn pups was not significantly different between the test groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study.


The sex distribution and sex ratios of live F2 pups on the day of birth and on PND 21 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.
Dose descriptor:
NOAEL
Remarks:
200 ppm
Generation:
F2
Effect level:
18 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: NOAEL for developmental toxicity in F2 generation
Reproductive effects observed:
yes
Lowest effective dose / conc.:
1 800 ppm
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Conclusions:
Overall, under the conditions of the present study the NOAEL for general, systemic toxicity is below 200 ppm (about 18 mg/kg bw/d) in the F0 parental rat, based on evidence for liver toxicity and corresponding thyroid histopathology and thyroid hormone changes in all test groups. The NOAEL in the F1 adult rats is 200 ppm. At 1800 ppm (about 167 mg/kg bw/d) distinct toxicity such as decreased body weight/body weight gain, anemia as well as liver and thyroid toxicity
was noted in the F0 parental animals as well as adolescent and adult F1 offspring, including F1B parental rats.
The NOAEL for fertility and reproductive performance for the F0 and F1 parental rats is 600 ppm (about 54 mg/kg bw/d), based on lower numbers of implants and subsequently smallerp litters at the LOAEL (Lowest Observed Adverse Effect Level) of 1800 ppm (about 167 mg/kg bw/d).
The NOAEL for developmental toxicity in the F1 and F2 progeny is 200 ppm (about 18 mg/kg bw/d), based on reduced preweaning body weight gain, which was observed at the LOAEL (Lowest Observed Adverse Effect Level) of 600 ppm (about 54 mg/kg bw/d).
The NOAEL for developmental neurotoxicity for the F1 progeny is 600 ppm (about 54 mg/kg bw/d), based on an increased incidence of focal to multifocal axonal degeneration in the white matter of thoracic cord, which was observed at the LOAEL (Lowest Observed Adverse Effect level) of 1800 ppm (about 167 mg/kg bw/d).
Effect on fertility: via oral route
Dose descriptor:
NOAEL
54 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The test substance was tested for reproductive toxicity in an OECD 422, an OECD 421 'plus' range finding, as well as an OECD 443 study. All studies were performed in compliance with GLP regulations.


 


In the OECD 422 combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, rats were treated with the test substance by oral gavage at dose levels of 25, 75 and 225 mg/kg bw/d. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 28 days). Females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 39-45 days). In parental animals hepatic toxicity was noted for animals of both sexes at the high dose of 225 mg/kg bw/d. This was supported by changes in clinical pathology endpoints (increase of ALP, bilirubin, cholesterol and decrease of albumin, total protein and inorganic phosphate, among others), macroscopic abnormalities (accentuated lobular pattern, enlargement and discoloration), increased liver weights (relative weights approximately 50% higher for males) and microscopic findings including hepatocellular vacuolation and hypertrophy. At 75 mg/kg bw/day, morphologic liver findings were regarded to be an adaptive, non-adverse response based on the minimal severity of the hypertrophy (correlated to pale discoloration) and low incidence and minimal degree of vacuolation in the females. However, when taken together with similar changes seen in clinical biochemistry parameters in high dose animals and an approximate relative liver weight increase of 24% and 17% for males and females, respectively, the findings were also considered toxicologically relevant at 75 mg/kg bw/day. Organ weight changes at 25 mg/kg bw/day were not considered toxicologically relevant as there were no corroborative changes in clinical biochemistry parameters indicative of liver damage. No treatment-related toxicologically significant changes were noted in any of the remaining parental parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption). Regarding reproductive toxicity, no toxicity was observed up to the highest dose level tested (225 mg/kg bw/day). Effects in developmental toxicity were seen as females at 225 mg/kg bw/day had a lower mean number of pups at the first litter check compared to controls (7.9 versus 10.9). An increase in postnatal loss and a correspondingly lower viability index were seen for females at this dose level (8 versus 0 in controls). While the majority of the postnatal loss was attributable to one female, other litters were also affected and a possible effect of treatment could not be excluded. No treatment-related changes were noted in any of the remaining developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development including, clinical signs, body weight and macroscopy). In conclusion, the treatment of male and female Wistar Han rats by oral gavage at dose levels of 25, 75 and 225 mg/kg revealed parental (hepatic) toxicity at 75 and 225 mg/kg bw/day. Developmental toxicity was seen at 225 mg/kg bw/day only. No reproductive toxicity was observed with treatment up to 225 mg/kg bw/day.


 


 


The range-finding study (OECD 421 plus) was performed prior to the OECD 443 study, with a 10 week-premating period. Dietary test substance concentrations of 300, 1000 and 3000 ppm were fed to 10 Wistar rats per dose and sex (concentrations were reduced to 50% during lactation). After the acclimatization period, the test substance was administered as addition to the diet continuously throughout the entire study. The duration of treatment covered a 10-week premating in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 13 days of lactation and roughly 2 weeks thereafter in females. The animals of the control group were treated in the same way with the vehicle only (diet). Treatment ended about 16 - 20 hours before sacrifice. Ten weeks after the beginning of treatment, the surviving males and females from the same test group were mated overnight in a ratio of 1:1 for a maximum of 2 weeks. On PND 4, the individual litters were standardized in such a way that, where possible, each litter contained 4 male and 4 female pups. Signs of general systemic toxicity were observed in all parental animals of the high dose group (3000 ppm) and in females of the intermediate dose group (1500 ppm) which showed alterations of food consumption and body weight (changes). Clinical pathology indicated as changes in liver metabolism as decreased total bile acid (TBA) levels in rats of both sexes of the 1000 and 3000 ppm test groups and increased triglyceride values in male and female rats of the high dose group as well as decreased albumin and increased cholesterol values in females of the high dose group. Higher activities of gamma-glutamyl transferase (GGT) in dams of the high dose group and increased alkaline phosphatase (ALP) activities in both sexes of the 1000 and 3000 ppm groups as well as in females of the low dose group (300 ppm) were most probably due to a liver enzyme induction coupled with a liver cell swelling. Most probably in consequence of the liver enzyme induction, T4 values in adult high dose group males were decreased because of accelerated clearance of the conjugated hormone via the bile. As feedback mechanism TSH values were increased in these individuals. Enzyme induction led to lower total bilirubin levels in high dose group males because of an increased bilirubin conjugation and accelerated excretion via the bile. In contrast, in dams of this test group total bilirubin values were increased, presumably because of a bile  congestion. Consistently, decreased total bile acid levels in rats of both sexes of intermediate and high dose groups (1000 and 3000 ppm) were most probably due to a decreased bile acid synthesis because of the changed liver cell metabolism. This was confirmed at least in high dose group females by lower albumin synthesis leading to lower serum levels of albumin. Pathological examination exhibited, the liver and the thyroid glands in males and females as target organs. The liver showed a significant absolute and relative weight increase in males and a  significant relative weight increase in intermediate and high dose group females. A minimal but significant relative weight increase was also noted in low dose group males. These weight changes were consistent with liver cell hypertrophies and fatty changes, which showed varying patterns at the different dose levels. A significant relative weight increase of the thyroid glands in high dose group males correlated with histopathological and hormonal changes. In females, a significant final body weight decrease was observed in the intermediate and high dose groups (-9% and -15%, respectively). At 3000 ppm histopathology showed a hepatocellular hypertrophy in the liver which was mainly diffuse with mild centrilobular accentuation (minimal to moderate) affecting all male and female animals. A periportal fatty change, mainly of microvesicular with some  macrovesicular type was observed in males (minimal) and females (minimal to slight). An additional focal necrosis was found in one male. All these changes associated with significant and relevant relative liver weight increases (males +43%, females +30%) were regarded as treatment related and adverse. In the thyroid glands of males, hypertrophy/hyperplasia of follicular cells showed a  increase in incidence (9 out of 10 males) and grading (minimal to moderate) accompanied by altered, flaky colloid, also increased in incidence and grading. These changes were regarded as treatment-related and adverse as they were consistent with altered hormonal values in the clinical chemistry (decreased T4 and increased THS, both statistically significant). In females, the hypertrophy/hyperplasia of follicular cells, also accompanied by altered colloid with flaky appearance, showed a lesser incidence and grading than in males (6 out of 10, minimal to slight) but it was also considered treatment-related. However, since thyroid gland hormones were not altered, the histopathological findings were assessed as treatment related but not adverse. At the intermediate dose of 1000 ppm, all males showed centrilobular  hepatocellular hypertrophy (minimal to slight), correlating with significant liver weight increases (absolute +12%, relative +14%). In addition, minimal fatty change (4 out of 10 males) of predominant macrovesicular type and midzone localization, together with minimal single cell necrosis/apoptosis (6 out of 10 males) and focal necrosis (one male out of 10) was observed. In females, 8 out of 10 showed a minimal centrilobular liver cell hypertrophy correlating with a significant relative weight increase (+16%). The thyroid glands of males showed minimal hypertrophy/hyperplasia of follicular cells (4 out of 10 males) accompanied by altered flaky colloid. These changes were assessed as treatment-related but not adverse due to the lack of hormonal changes. In females, a minimal hypertrophy/hyperplasia of follicular cells (2 out of 10), although no clearly dose-dependent, was associated with the presence of altered flaky colloid and was assumed to be possibly treatment-related but not adverse. In the low dose group (300 ppm), 2 out of 10 male animals displayed centrilobular hepatocellular hypertrophy (minimal and slight). Furthermore, minimal single cell necrosis/apoptosis was present in these two male animals. All these findings were considered treatment-related and potentially adverse. No treatment-related findings were observed in female animals. In the thyroid gland, the incidence of hypertrophy/hyperplasia of follicular cells in males (3 out of 10) and females (3 out of 10) were minimally above the historical control data and therefore, they were assumed as possibly treatment-related but not adverse. Effects on fertility and reproductive performance were seen in the high dose group (3000 ppm). A treatment related increase of estrous cycle length as well as decreased number of implantations sites and pups delivered. No signs of reproductive toxicity were observed in male or female parental animals of the 300, and 1000 ppm groups. Developmental toxicity was observed in the high dose group as decreased body weight gain was observed in male and female pups starting from after birth and resulting in decreased body weight from postnatal day (PND) 7 onwards. The deviations from control values were increasing over time. In these male pups the number of areola/nipples and the nipple number per animal were higher. Since the sexual maturation is related to the body weight development (Melching-Kollmuss et al 2017), this observation is at least partially related to the delay of general development in male pups. Furthermore, a significant lower number of pregnant rats was observed without dose-dependency in the lowest dose group (300 ppm), which was not assessed as treatment-related. Under the conditions of this Reproduction/Developmental Toxicity Screening Test in Wistar Rats, the oral administration of Benzenamine, N-phenyl-, reaction products with 2,4,4- trimethylpentene to male and female Wistar rats via the diet revealed signs of systemic toxicity at all concentrations tested from 300 to 3000 ppm. Based on altered liver parameters in clinical pathology and pathology, the LOAEL for general systemic toxicity is 300 ppm for male (26 mg/kg bw/d) and female Wistar rats (28 mg/kg bw/d). The NOAEL for reproductive performance and fertility is 1000 ppm for male (87 mg/kg bw/d) and female (95 mg/kg bw/d) Wistar rats. The NOAEL for developmental toxicity is 1000 ppm (95 mg/kg bw/d) in parental females.


 


 


In the OECD 443 guideline study, the test substance was administered to groups of 25 male and 25 female young Wistar rats as a homogeneous addition to the food in different concentrations (0, 200, 600 and 1800 ppm). F0 animals were treated at least for 10 weeks prior to mating to produce one litter (F1 generation). Pups of the F1 litter were selected (F1 rearing animals) and assigned to 4 different cohorts (1A, 1B, 2A and 2B) which were subjected to specific postweaning examinations. Cohort 1B (=F1 generation parental animals) was selected to produce F2 pups. Cohort 2A and 2B animals were assigned as developmental neurotoxicity cohorts. Cohort 2A animals were sacrificed after 11 and cohort 2B animals after 3 weeks. F1 animals selected for breeding were continued in the same dose group as their parents. Groups of 25 males and 25 females, selected from F1 pups to become F1 parental generation, were offered diets containing target dosages of 0, 200, 600 and 1800 ppm of the test substance post weaning, and the breeding program was repeated to produce a F2 litter. The study was terminated with the terminal sacrifice of the F1 rearing animals of cohort 1B. Test diets containing the test substance were offered continuously throughout the study. During the lactation period the test substance concentrations in the diet of the F0 and F1 females were reduced to 50%. The general state of health of parental animals and the pups was checked each day, and parental animals were examined for their mating and reproductive performances. Food consumption of the F0 parents and F1 rearing animals was determined regularly once per week (except for food consumption of the F0 and F1B males which was determined after the 10th premating week) and weekly for F0 and F1B females during gestation days (GD) 0-7, 7-14, 14-20 and postnatal days (PND) 1-4, 4-7, 7-10, 10-14, 14-18 and 18-21. In general, body weights of F0 parents and F1 rearing animals were determined once weekly. However, during gestation and lactation F0 and F1B females were weighed on GD 0, 7, 14 and 20 and on PND 1, 4, 7, 10, 14, 18 and 21. A detailed clinical observation was performed in all F0 parents and F1 animals in cohorts 1A, 1B and 2A at weekly intervals. Estrous cycle data were evaluated for F0 and F1B females over a three week period prior to mating until evidence of mating occurred. In all cohort 1A females, vaginal smears were collected after vaginal opening until the first cornified smear (estrous) was recorded. The estrous cycle also was evaluated in cohort 1A females for 2 weeks around PND 75. Moreover, the estrous stage of each F0, 1A and 1B female was determined on the day of scheduled sacrifice. The F1 and F2 pups were sexed on the day of birth (PND 0) and were weighed on the first day after birth (PND 1) as well as on PND 4, 7, 14 and 21. At necropsy, all pups were examined macroscopically (including weight determinations of brain, spleen and thymus in one pup/sex/litter of F2 pups). Anogenital distance measurements were conducted in a blind randomized fashion on all live male and female pups on PND 1. All surviving pups were examined for the presence or absence of nipple/areola anlagen on PND 13 and were re-examined on PND 20. If nipple/areola anlagen were recorded, all surviving male pups were carefully re-examined one day prior to necropsy. The number of nipple/areola anlagen was counted. Date of sexual maturation, i.e. day of vaginal opening or balanopreputial separation of all F1 pups selected to become F1 rearing animals (except F2B rearing animals) was recorded. Urine samples for clinical pathological investigations were withdrawn from 10 selected F0 and cohort 1A animals per sex and group. Blood samples for clinical pathological investigations were withdrawn from 10 selected F0 and cohort 1A animals per sex and group. Further blood samples were taken from all surplus (culled) PND 4 pups per sex and group as well as from 10 surplus PND 22 pups per sex and group. Various sperm parameters (motility, sperm head count, morphology) were assessed in the F0 generation males and cohort 1A males at scheduled sacrifice or after appropriate staining. All F0 and F1B parental animals were assessed by gross pathology (including weight determinations of several organs) and subjected to an extensive histopathological examination; special attention being paid to the organs of the reproductive system. A quantitative assessment of primordial and growing follicles in the ovaries was performed for all control and high-dose F1 rearing females of cohort 1A. All F1 rearing animals were assessed by different pathological, neuro- and histopathological examinations. Concentration and homogeneity analysis of treated feed verified the homogeneity and confirmed correct target concentrations of the test substance within the feed. The overall mean doses throughout all study phases and across all cohorts for males were approx. 18 mg/kg bw/d in the 200 ppm group, approx. 54 mg/kg bw/d in the 600 ppm group and approx. 167 mg/kg bw/d in the 1800 ppm group and for females approx. 18 mg/kg bw/d in the 200 ppm group, approx. 54 mg/kg bw/d in the 600 ppm group and approx. 166 mg/kg bw/d in the 1800 ppm group. None of the tested groups showed treatment related clinical signs or mortality. The body weight and body weight change of 1800 ppm female F0 rats was below control values during major parts of premating as well as gestation and lactation periods. Although male animals of this dose level showed frequent episodes of decreased body weight gain body weights of these animals were not affected. In F1 animals of both sexes treated with 1800 ppm, body weights and body weight change were consistently below the control throughout the in-life period. Particularly, high dose group F0 and F1B dams showed weight reductions during their gestation and lactation periods. F0 and F1 animals of the 200 and 600 ppm dose groups, also showed episodes of drecreased body weight gain predominantly in males but also some in females. Magnitude and frequency of these changes was generally lower than at 1800 ppm but it may still represent a treatment related effect. Food consumption in the 1800 ppm F0 and F1B dams food consumption was consistently reduced during gestation and lactation. Additionally, reductions were seen in the F1B females throughout premating period. In the 200 ppm and 600 ppm dose group F0 males and females as well as in F1 adolescents (Cohort 1A, 1B and 2A) of all dose groups, the food consumption remained either unchanged or only showed episodes of inconsistent changes.


In clinical pathology of high dose group parental F0 male and female rats  decreased hemoglobin and hematocrit values indicated an anemia. In males higher reticulocyte counts were indicative of a regenerative anemia. In the liver an increased synthesis of coagulation factors resulted in a reduced prothrombin time, higher triglyceride and lower albumin levels in rats of both sexes as well as lower total protein values in males of this test group. In females, this was accompanied by increased activities of γ-glutamyl transferase (GGT) activities as well as higher globulin and cholesterol values. Higher alkaline phosphatase (ALP) activities in male and female rats receiving 1800 ppm were also due to liver enzyme induction. Furthermore, an increased alanine aminotransferase (ALT) in males of this test group was observed. The altered coagulation factor synthesis led to decreased platelet counts in females. Higher ALP activities and triglyceride values were already observed in male and female rats of the 600 ppm test group. In high dose group F1 cohort 1A rats (1800 ppm) similar clinical pathology changes were observed at study day 90. At the intermediate dose level of 600 ppm in F1A male and female rats increased ALP activities and lower albumin levels, with lower total protein and globulin levels in males were seen. Increases of ALP activities and decreases of albumin levels were observed already in low dose group females (200 ppm).


In pathology treatment-related changes were seen in the liver and subsequently in the thyroid in F0 and F1 males and females. In the F0 parental generation, intermediate and high dose group males and females (600 and 1800 ppm) showed increased liver weight which was caused by a diffuse or centrilobular hepatocellular liver cell hypertrophy and in males by an increase of fat in the midzonal-centrilobular region. In combination with changed parameters in clinical pathology  these findings were considered as treatment-related and adverse. A minimal centrilobular fat storage in two male animals and a minimal hepatocellular centrilobular hypertrophy in seven females were already seen at the low dose which was regarded as treatment related but not adverse. F1 cohort 1A females of the intermediate and high dose groups (600 and 1800 ppm) showed increased liver weight which corresponded to a centrilobular liver cell hypertrophy. In the same test groups males revealed an increase in centrilobular hypertrophy and fatty change. In combination with findings in clinical pathology, the weight changes and liver cell hypertrophy were regarded to be treatment-related and adverse. In the F1 cohort 1A low dose group (200 ppm) two males revealed centrilobular hypertrophy. As clinical pathology parameters were not changed, this finding might be treatment-related but not as adverse. In F1 cohort 1B, high dose group male animals (1800 ppm) showed an increase in relative liver weight and females of the same test group an increase of absolute and relative weight. Other findings in this cohort occurred either individually or were biologically equally distributed over control and treatment groups.


In the thyroid glands of F0 males and females of all test groups  hypertrophy/hyperplasia of follicular cells and altered colloid were observed, which corresponded to increased organ weights in the high dose group. Because there were also changes in hormone levels observed in clinical pathology, these findings were regarded to be treatment-related and adverse. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. Regarding F1 cohort 1A animals, the thyroid glands of high dose group males and females of the low and high dose groups (600 and 1800 ppm) showed follicular cell hypertrophy/hyperplasia. Altered colloid was only seen in male and female animals of the high dose group (1800 ppm). In combination with clinical pathology findings in these test groups, the finding was regarded to be treatment-related and adverse. Because further findings in the thyroid glands of intermediate and low dose group males did not show a dose-response relationship and lacked findings in clinical pathology they were considered as incidental and unrelated to treatment.


Measurement of T4 and TSH thyroid hormones revealed effects in the F0 and F1 adult animals. No effects were observed in the F1 and F2 preweaning offspring. In F0 parental rats, T4 decreases were observed in males and TSH increases were found in both sexes of all dose groups. Regarding the findings made in liver parameters, it can be assumed that the thyroid hormone level changes happened secondary to a liver cell enzyme induction because of an accelerated clearance of T4 via the liver coupled with an enhanced T4 synthesis induced by higher TSH releases via an endocrine feedback mechanism. It also seems that this feedback maintains the serum T4 homeostasis in parental females, but not in F0 males. In high dose group F1 cohort 1A animals of both sexes TSH values were increased, but T4 values were decreased only in males of this test group. This corresponds to the thyroid hormone changes mentioned in F0 rats. The  mechanism of increased clearance of T4 via the liver is supported by the fact that exclusively the grown-up F1 rats showed thyroid hormone changes, because liver cell enzyme activity may not be fully established in the pups until PND 22. In F1A females of the intermediate dose group (600 ppm) T4 values were significantly decreased and TSH values were increased. Although T4 decreases were not dose dependent, these findings correlate with the histopathological observations in the thyroids. 


There were no indications from clinical examinations as well as gross and histopathology, that treatment adversely affected the fertility or reproductive performance of the F0 and F1B parental animals up to and including the administered intermediate dose of 600 ppm. Estrous cycle data, sperm quality of males, mating behavior, conception, gestation, parturition, lactation and weaning as well as sexual organ weights and gross and histopathological findings of these organs (specifically the differential ovarian follicle count) were comparable between the rats of low- and mid-dose and control groups and ranged within the historical control data of the test facility.


At a dose of 1800 ppm significantly reduced numbers of implants were observed, which were below the range of historical controls. This subsequently caused a smaller average litter size in F1 and F2 offspring of the high dose group. However, because estrous cycle data, sperm quality of males, mating behavior, conception, gestation, parturition, lactation and weaning as well as sexual organ weights and morphology of these organs were unchanged in the F0 and F1B parents of this offspring, it is likely that the considerable stress caused by maternal toxicity at the high dose, which was particularly distinct during gestation/lactation, is related to the lower implantation success.


Pup body weight development was affected in high dose group offspring (1800 ppm) approximately from day 7 of lactation. The offspring weighed less than controls during the course of lactation and their weights were below the historical control range. There was no treatment-related influence on high dose F1 and F2 pup body weight change at birth and shortly thereafter. In the last two weeks of lactation (PND 7 - 21) the mean body weight change of these pups was significantly below control values. The F1 intermediate dose offspring (600 ppm) was similarly affected, but to a lower extent. The F2 intermediate dose level offspring remained unaffected. During the last week of lactation the offspring already consumed considerable amounts of treated diet and the post-weaning body weight gain of the high dose adolescents in the various F1 cohorts continued to be lower compared to the controls. Therefore, it was assumed that the lower pre-weaning body weight/body weight gain at 600 and 1800 ppm was caused by direct exposure of the offspring to the test compound through the diet as much as by developmental toxicity. However, the pup body weight effects had no influence on postnatal pup survival or well-being, neither during early lactation nor later, as clinical and/or gross necropsy examinations of the mid and high dose F1 and F2 pups revealed no adverse findings.


Analysis of the anogenital distance/index and the presence of nipples/areolas revealed no findings, indicating the absence of endocrine-mediated imbalances. However, there was no difference in the percentage of F2 male pups having nipples/areolae but the mean nipple number per pup was statistically significantly above the concurrent control values in the 1800 ppm F2 male pups when examined on PND 13. It should be noted thtat this is considered to be the consequence of a general delay of pup development, rather than a specific effect on hormonal homeostasis. Furthermore, no nipples/areolae were detected in any male pup of all test groups during the re-examination on PND 20.


Regarding developmental landmarks, a statistically significant delay in vaginal opening of less than one day beyond the concurrent control was observed in female F1 offspring of the high dose group (1800 ppm). However, the delay
is within the historical control range of the test facility and there was a consistent effect on postweaning body weight development in the affected females notable, and no corresponding increase in body weight was observed in the older animals at the time of puberty. Furthermore, there was no effect on estrous cyclicity or the integrity of sexual organs in these females, including differential ovarian follicle count, during later life. Similarly, there was a statistically significant delay in preputial separation of about 1.5 days, which exceeded the control data in male F1 offspring of the high dose group. The delay is within the historical control range of the test facility. Again, there was a consistent effect on postweaning body weight development in the affected males notable and no corresponding increase in body weight was observed in the older animals at the time of puberty. In addition, no effect on the integrity of sexual organs (including accessory sexual glands) was noted in all treated males. Hence, it was assumed that this rather small delay in onset of puberty in both sexes was most likely a consequence of systemic toxicity and subsequent general developmental delay, and not a specific effect on the timing of puberty. Furthermore, there was no evidence that the test substance impaired development of neuronal function in the F1 offspring as demonstrated by the absence of relevant effects in a functional observation battery (FOB) as well as automated motor activity and auditory startle. However, in neurohistopathology of cohort 2A animals, which were sacrificed at PND 77 (includes post-weaning), an increased incidence of focal to multifocal axonal degeneration in the white matter characterized by digestion chambers with occasional pyknotic nuclei and presence of gitter cells was observed in male animals of the high dose group. No  corresponding analysis for this specific finding was performed in cohort 2B animals, which were sacrificed on PND 22. Because it is not possible based on this data to differentiate wether this effect is a chronic toxicity effect or rather a developmental toxicity effect, the respective analysis of cohort 2B (PND 22) animals is scheduled for analysis.


Overall, the NOAEL for general, systemic toxicity in this study is below 200
ppm (about 18 mg/kg bw/d) in the F0 parental rat, based on evidence for liver toxicity and corresponding secondary thyroid histopathology and thyroid hormone changes in all test groups. In F1 adult rats, the NOAEL is 200 ppm. At 1800 ppm (about 167 mg/kg bw/d) distinct toxicity such as decreased body weight/body weight gain, anemia as well as liver and subsequent thyroid toxicity was noted in the F0 parental animals as well as adolescent and adult F1 offspring, including
F1B parental rats.
The NOAEL for fertility and reproductive performance for the F0 and F1 parental rats is 600 ppm (about 54 mg/kg bw/d), based on lower numbers of implants and subsequently smaller litters, in the presence of maternal toxicity.


The NOAEL for developmental toxicity in the F1 and F2 progeny is 200 ppm (about 18 mg/kg bw/d), based on reduced preweaning body weight gain, which was presumably caused by direct exposure of the offspring to the test compound through the diet as much as by developmental toxicity.


Regarding findings in the spinal cord of cohort 2A animals, further tests have been scheduled to be able to make a final conclusion wether the findings in neurohistopathology should be regarded as developmental effects or rather as  chronic toxicity effects.


 

Effects on developmental toxicity

Description of key information

Information on developmental toxicity is available from an OECD 422, as well as an OECD 443 study.


 


OECD 422:Combined 28-day repeated dose toxicity study with the reproductin/developmental toxicity screening test in rats by oral gavage:


Dosages: 25, 75, 225 mg/kg bw/d


Parental NOAEL: 25 mg/kg bw/day:


Hepatic toxicity in animals of both sexes at 225 mg/kg bw/day. At 75 mg/kg bw/d, morphologic liver findings were regarded to be an adaptive, non-adverse response. However, additional changes at 75 mg/kg bw/d in clinical biochemistry and a relative liver weight increase were considered toxicologically relevant.


 


Developmental NOAEL: 75 mg/kg bw/day:


Females at 225 mg/kg bw/day had a lower mean number of pups at the first litter check. An increase in postnatal loss and a correspondingly lower viability index
were seen for females at this dose level.


 


OECD 443: Extended One-Generation Reproduction Toxicity Study in Wistar Rats Administration via the Diet:


Dosages: 200, 600, 1800 ppm. With 10 weeks premating, extension of cohort 1B and developmental neurotoxicity cohorts 2A and 2B.


General, systemic toxicity LOAEL F0: approx. 18 mg/kg bw/d (200 ppm):


Evidence for liver toxicity and corresponding thyroid histopathology and thyroid hormone changes in all test groups


 


General, systemic toxicity NOAEL F1: approx. 18 mg/kg bw/d (200ppm):


Distinct toxicity such as decreased body weight/body weight gain, anemia as well as liver and thyroid toxicity.


 


Develeopmental toxicity NOAEL in F1 and F2: Approx. 18 mg/kg bw/d (200ppm):


Reduced preweaning body weight gain.


 


Develeopmental neurotoxicity NOAEL in F1: Approx. 54 mg/kg bw/d (200ppm):


Increased incidence of focal to multifocal axonal degeneration in the white matter of thoracic cord. However, no clear distinction can be made wether this effect is a developmental or a chronic toxicity effect. Therefore, further analysis has already been initialized.


 


 

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study itself is vaild, but it is only a screening study for developmental toxicity.
Qualifier:
according to guideline
Guideline:
other: OECD 422
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): Benzenamine, N-phenyl-,reaction products with 2,4,4-trimethylpentene
- Substance type: UVCB
- Physical state: Clear slightly yellow viscous liquid
- Analytical purity: 100% UVCB
- Purity test date: 2014 , study number 13L00223
- Lot/batch No.: 40401913D
- Expiration date of the lot/batch: 09 February 2016
- Stability under test conditions: stable
- Storage condition of test material: At room temperature in the dark

Test substance handling: Avoid temperatures <10°C, maximum temperature:30°C. In the case of crystallization, heat up the substance (temperature up to 30°C) until a clear solution is obtained
Specific Gravity / Density 0.975 g/cm3 (20°C)
pH 5.1-6.2 (1%(m), 20-25°C) (as suspension)
Stability at higher temperatures Maximum temperature: 30°C
Species:
rat
Strain:
other: Crl:WI(Han)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 10-12 weeks
- Weight at study initiation: 353 g (males), 210 g (females)
- Fasting period before study: none
- Housing: groups of 5 animals/sex/cage (premating males and females and postmating males), otherwise single cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%,
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 13 March 2014 To: 1 May 2014
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) are be prepared daily within 6 hours prior to dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Test item is insoluble in water, but soluble in corn oil
- Amount of vehicle (if gavage): 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
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
90-110% 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
- Length of cohabitation: 14 days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy
Duration of treatment / exposure:
28 days (males), ca 53 days (females)
Frequency of treatment:
daily
Duration of test:
28 days (males), ca 53 days (females)
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Dose / conc.:
225 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
5 animals/sex/group were selected for functional observations, locomotor activity, clinical pathology, organ weights and histopathology
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Range-finding standy
- Rationale for animal assignment (if not random): Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
The substance caused adverse effects on liver of males and females at dose levels of 75 and 225 mg/kg bw. For details it is referred to the endpoint study records of repeated dose toxicity in IUCLID chapter 7.5.1.
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Abnormalities:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Postnatal loss was increased (8 pups in total, versus 0 pups in the control group), but 5 of the 8 pups all came from one dam.
There were not adverse effects regarding other developmental toxicity endpoints at any dose group (eg pup body weight, clinical signs, macroscopic investigation, necropsy).
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
changes in postnatal survival
Remarks on result:
other: Increase in postnatal pup mortality at 225 mg/kg bw., mostly in one animal and unlikely to be a true effect
Abnormalities:
not examined
Developmental effects observed:
yes
Lowest effective dose / conc.:
225 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Dose-range-finding study, therefore limited examinations
Principles of method if other than guideline:
Dose-range-finding study in pregnant rats
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia S.p.A.
- Age at study initiation: 10 weeks
- Weight at study initiation: 176-200 g
- Fasting period before study: none
- Housing: single cages (after mating)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 18 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C2 °C
- Humidity (%): 55%15%
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12/+2

IN-LIFE DATES: From: 2014-03-25 To: 2014-04-20
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The substance was suspended in corn oil and brought to the final volume
appropriate for each concentration (75 and 250 mg/mL).

VEHICLE
- Justification for use and choice of vehicle (if other than water): The substance is not soluble or dispersible in water. It is miscible in corn oil.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulation analyses were carried out by the Analytical Chemistry Department at the testing facility according to a validated method including the assessment of stability up to 24 hours at room temperature.
Prior to commencement of treatment, analysis was performed to confirm that the proposed formulation procedure was acceptable (content check and homogeneity). In addition, samples of the formulations, prepared on weeks 1 and 2 of treatment, were also analysed to measure the concentration and homogeneity. The results of analyses were within the acceptance ranges (90-110%)
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused: 1 male to 1 female
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
Gestation day 6-19
Frequency of treatment:
daily
Duration of test:
14 days (GD 6-necropsy on GD20)
Remarks:
Doses / Concentrations:
300 and 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Dose-range finding study for OECD 414
Maternal examinations:
Body weight
Food consumption
Clincial chemistry and haematology (see list below)
Organ weights:
– Liver
– Kidneys
– Adrenals
– Spleen
Statistics:
yes
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
No treatment-related mortality was observed.
No clinical signs were recorded through the study with the exception of one high dose female which showed piloerection on gestation Day 20.

Significantly reduced body weight was found in the high dose group (1000 mg/kg bw, -10%) starting from Day 9 of gestation up to the end of study when compared to controls. Body weight gain was also reduced in the high dose group respect to the control group and this difference was statistically significant on Days 9 and 12 of gestation.

Food consumption was significantly reduced in the high dose group ( 66% of control values) when compared to the control group from gestation Day 9 until the end of study.


When compared with controls, some animals dosed with 300 and 1000 mg/kg/day showed higher values of activated partial thromboplastin time. Mean group values were 15% to 23% above controls.
No further changes of toxicological significance were recorded for haematolgoy.

Fluctuations of liver/metabolic markers were recorded in treated animals, mainly those receiving 1000 mg/kg/day. In particular, these animals showed
increases of alkaline phosphatase, alanine aminotransferase, cholesterol, triglycerides, glucose and decreases of creatinine, protein, albumin, globulin, calcium and bile acids. Some of the above changes were also recorded in animals dosed with 300 mg/kg/day including increases of alkaline phosphatase, triglycerides and decrease of albumin and bile acids.

Terminal body weight, corrected body weight (body weight at necropsy minus gravid uterus weight) and corrected body weight gain (body weight
at necropsy minus gravid uterus weight, minus body weight at Day 6 of pregnancy) were significantly reduced in the high dose group. No differences
were found in the uterus weight between groups.

Changes in absolute and/or relative organ weights were found in the high dose group which included increased absolute and relative adrenals weights and
decreased absolute spleen and kidneys weights when compared to controls.
Relative liver weight was instead significantly increased in the low and high dose groups.

Pale discoloration of liver was observed in four animals of the high dose group.

Based on the effects observed on liver and body weight gain, 500 mg/kg bw is chosen as high dose for the actual OECD 414 study. This dose is expected to cause some, but not excessive maternal toxicity.
Dose descriptor:
LOEL
Effect level:
300 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:not examined

Details on embryotoxic / teratogenic effects:
All pregnant females had live fetuses.
Abnormalities:
not specified
Developmental effects observed:
not specified

In the control and high dose group, each one dam was not pregnant. Mean values were only calculated for pregnant animals.

Conclusions:
Treatment of pregnant rats with doses of 300 and 1000 mg/kg bw via gavage caused adverse effects on liver.
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: OECD 414 and GLP compliant study. A default reliability of 2 is assigned for read-across according to ECHA guidance.
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Age at study initiation: (age at delivery 10 weeks)
- Weight at study initiation: (weight range at delivery 177-196 g)
- Fasting period before study: none
- Housing: individual cages (during gestation)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 18 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 °C
- Humidity (%): 55% +/- 15%
- Air changes (per hr): 15 -20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2014-05-20 To: 2014-06-16
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The formulations were prepared daily and the concentrations were calculated and expressed in terms of test item as supplied.


VEHICLE
- Justification for use and choice of vehicle: The substance is miscible in corn oil and insoluble in water.
- Concentration in vehicle: 12.5, 37.5 and 125 mg/mL
- Amount of vehicle: 4 ml/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The proposed formulation procedure for the test item was checked in the range from 12.5 to 125 mg/mL by chemical analysis (concentration and
homogeneity) during the pre-treatment period to confirm that the method was suitable. Final results for all levels were within the acceptability limits
for concentration (90-110%). Stability after 24 hours at room temperature was verified in the range from 1 to 300 mg/mL in the validation study.
Samples of the formulations prepared on week 1 and Last Week were analysed to check the homogeneity and concentration.
Details on mating procedure:
Females were paired one to one in the home cage of the male and left overnight. Vaginal smears were taken daily in the morning from the day after pairing until a positive identification of matingwas made. The day of mating, as judged by the presence of sperm in the vaginal smear or by the presence of a copulation plug, was considered as Day 0 of gestation (or Day 0 post coitum).
Duration of treatment / exposure:
Day 6 through Day 19 post coitum
Frequency of treatment:
daily
Duration of test:
Day 6 through Day 20 post coitum
Remarks:
Doses / Concentrations:
50, 150 and 500 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Range-finding study with pregnant rats. The highest dose group of 500 mg/kg was expected to cause maternal toxicity as indicated by adverse effects on body weights and evidence on liver toxicity as indicated by clinical chemistry parameters. For details it is referred to the robust study summary of the maternal toxicity study.
- Rationale for animal assignment: Females were allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 3, 6, 9, 12, 15, 18 and 20 post coitum

FOOD CONSUMPTION : Yes
- Time schedule for examinations: Days 3, 6, 9, 12, 15, 18 and 20 post coitum starting from Day 0 post coitum


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: ovaries and uteri

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: gross evaluation of placentae, number of intra-uterine deaths,. Uteri or individual uterine horns without visible implantations were immersed
in a 20% solution of ammonium sulphide to reveal evidence of embryonic death at very early stages of implantation.
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
Statistics:
For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t-test, depending on the
homogeneity of data. Statistical analysis of non-continuous variables was carried out by means of the Kruskal-Wallis test and intergroup differences
between the control and treated groups assessed by a non-parametric version of the Williams test. The mean values, standard deviations and statistical
analysis were calculated from actual values in the computer without rounding off.
Indices:
Preimplantation loss
Postimplantation loss
Total implantation loss
Sex ratios
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
A slight decrease in body weight was noted in all treated females when compared to controls, reaching a statistical significance in females receiving
500 mg/kg bw/day (up to 7%), starting from Day 9 post coitum until the end of the study.
Statistically significant decrease was also recorded in body weight gain of females of the same group on Day 9 post coitum (109%; body weight loss) and Day 12 post coitum (22%). Starting from Day 15 post coitum the mean values of body weight gain were comparable between control and high dose group.

Statistically significant decrease (up to 22%) in food consumption was observed in treated females receiving 500 mg/kg bw/day, starting from Day 9 post coitum until the end of the study.

A slight trend to decrease was observed in terminal body weight of all treated females with respect to the control. This change was about -6% in the high
dose group, without statistical significance. A statistically significant decrease in corrected body weight (up to 6%) and corrected body weight gain (up
to 50%) was noted in treated females receiving 150 and 500 mg/kg bw/day. Gravid uterus weight was similar between control and treated groups.

There were no adverse findings at the macroscopic examination at necropsy.
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Abnormalities:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Litter data, mean foetal weight and sex ratio were unaffected by treatment.

A total of 15 small foetuses (foetal weight < 2.7 g) were detected: 2 out of 269 in the control group, 2 out of 214 in the low dose group, 1 out of 269 in the mid-dose group and 10 out of 254 in the high dose group. One foetus in the high dose group showed malrotation of the hindlimb, considered incidental. Of the ten small fetuses in the high dose group, 7 were from the dam which suffered most strongly from maternal toxicity as indicated by the lowest corrected body weight gain of minus 9.8g. This dam was also the only dam showing hunched posture and piloerection on gestation day 20. The higher incidence of small foetuses is therefore considered to be related to the lower maternal body weight gain.

No relevant findings that could be considered treatment-related were observed at visceral examination of foetuses in the treated groups, compared to controls.
The alterations recorded at skeletal examinations of foetuses were noted both in control and treated groups with a similar incidence.
Dose descriptor:
NOAEL
Effect level:
>= 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: teratogenicity
Remarks on result:
other: No teratogenicity at the hightest tested dose
Dose descriptor:
NOAEL
Effect level:
>= 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: embryotoxicity
Remarks on result:
other: No embrotoxicity at the highest tested dose
Abnormalities:
no effects observed
Developmental effects observed:
no

Table 1: TERMINAL BODY WEIGHT, UTERUS WEIGHT, CORRECTED BODY WEIGHT AND CORRECTED BODY WEIGHT GAIN OF FEMALES - GROUP MEAN DATA

Group   Terminal body weight (g) Gravid uterus weight (g) Body weight at necropsy minus gravid uterus weight (corrected body weight) (g)  Body weight at necropsy minus gravid uterus weight, minus body weight on GD6 (corrected body weight gain) (g) 
1 (control) Mean 336.38 66.40 269.98 29.87
  SD 15.84 8.80 14.79 7.67
  (n) 23 23 23 23
2 Me 323.9 60.42 263.5 29.45
  SD 23 18 18 16
  (n) 20 20 20 20
3 Me 323.8 66.20 257.6* 20.5*
  SD 28 12 18 9.
  (n) 23 23 23 23
4 Me 315.9 62.53 253.3* 14.83*
  SD 24 14 23 16
  (n) 22 22 22  22

* = Statistically significantly different from control group value at p< 0.05

Table 2: FOOD CONSUMPTION (g/animal/day) - GROUP MEAN

Group   gestation day 3 gestation day 6 gestation day 9 gestation day 12 gestation day 15 gestation day 18 gestation day 20
1 (control) n  23 23 23 23 23 23 23
  Mean 18.13 20.66 18.72 20.99 21.78 23.68 23.06
  SD 2.37 2.54 2.05 1.98 2.10 2.20 1.99
2 n  20 20 20 20 20 20 20
  Mean 17.99 20.97 18.32 20.64 21.04 23.43 22.83
  SD 2.3 3.03 2.46 2.19 2.68 2.99 4.33
3 n  23 23 23 23 23 23 23
  Mean 18.7 20.23 17.36 19.28 20.90 22.32 21.56
  SD 1.99 2.53 2.59 2.90 2.40 2.91 3.14
4 n  22 22.00 22 22.00 22.00 22.00 22.00
  Mean 18.24 20.97 15.33** 17.1** 19.01** 20.12** 18.02**
  SD 2.62 2.55 1.98 2.76 2.68 3.02 2.96

** = mean value of group is significantly different from control at p < 0.01

Table 3: Pregnancy status overview

Group 1 (control) 2 3 4
Initial group size (n) 24 24 24 24
Not pregnant (n) 1 4 1 2
Unilateral implantation (n) 0 1 0 0
With live foetuses at gestation Day 20 (n) 23 20 23 22

Table 4: Litter data and sex ratios - group mean data

    Corpus lutea Implantations Early Uterine Deaths Late Uterine Deaths Total Uterine Deaths Viable Young (total) Viable males Viable females % Males Preimplantation loss (%) Postimplantation loss Total implantation loss(%) Litter weight (g) Mean fetal weight (g)
1 Mean 12.87 12.26 0.52 0.04 0.57 11.7 6 5.7 50.96 4.68 4.15 8.52 43.1 3.69
  SD 1.63 1.63 1.47 0.21 1.5 1.77 2.24 2.05 17.27 5.13 10.27 11.78 6.71 0.34
  (n) 23 23 23 23 23 23 23 23 23 23 23 23 23 23
2 Mean 12.26 11.47 0.21 0.05 0.26 11.21 5.67 5.84 49.82 6.56 4.12 10.05 39.88 3.63
  SD 2.58 2.44 0.54 0.23 0.56 2.84 2.22 2.29 14.18 6.13 11.67 13.68 9.32 0.4
  (n) 19 19 19 19 19 19 18 19 18 19 19 19 19 19
3 Mean 13.09 12.22 0.52 0 0.52 11.7 5.78 5.91 449.19 6.28 4.73 10.86 42.41 3.65
  SD 2.7 2.35 0.73 0 0.73 2.55 1.93 1.98 12.79 6.68 6.74 7.39 8.85 0.28
  (n) 23 23 23 23 23 23 23 23 23 23 23 23 23 23
4 Mean 12.27 11.73 0.14 0 0.14 11.59 5.36 6.23 46.95 6.11 1.18 7.23 40.11 3.51
  SD 2.66 3.06 0.35 0 0.35 3.08 2.06 2.25 12.97 10.6 3.07 10.96 9.86 0.39
  (n) 22 22 22 22 22 22 22 22 22 22 22 22 22 22
Conclusions:
The substance is not teratogenic and not embryotoxic in rats. It causes maternal toxicity at a dose level of 500 mg/kg bw.
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-03-28 until
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
22 Jan 2001
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt fuer Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Strasse 7, 55116 Mainz, Germany
Species:
rabbit
Strain:
New Zealand White
Remarks:
Crl:KBL(NZW)
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% CMC suspension in deionized water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- specific amount of the test substance was weighed, topped up with vehicle (with 10 mg/100 mL Cremophor EL) in a calibrated beaker and intensely mixed with a magnetic stirrer
- test substance preparations were warmed up to approximately 30 degrees Celsius
- before and during administration, the preparations were kept homogeneous with a magnetic stirrer and kept at approximately 30 degrees Celsius with a heating sleeve or with a magnetic stirrer with heating plate

VEHICLE
- Justification for use and choice of vehicle: stability and homogenity could be proven in the vehicle
- Amount of vehicle: the preparation was administered as a 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
HPLC method with UV/VIS detection
Details on mating procedure:
- Impregnation procedure: artificial insemination
- GD0: day of insemination
Duration of treatment / exposure:
gestation days 6 through 28
Frequency of treatment:
daily
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: futher details are given in the dose-range finder in the the supporting study record.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily and if effects were observed, several times daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: (during GD 6-28) daily check, as well as within 5 hours after the administration

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 2, 4, 6, 9, 11, 14, 16, 19, 21, 23, 25, 28 and 29

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: uteri, ovaries
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened)
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter
Statistics:
- Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means: Food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight
- Pairwise comparison of each dose group with the control group using FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions: Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings
- Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians: Proportions of fetuses with malformations, variations and/or unclassified observations in each litter
Indices:
conception rate = number of pregnant animals / number of fertilized animals * 100
preimplantation loss, for each individual pregnant animal which underwent scheduled sacrifice = (number of corpora lutea – number of implantations) / number of corpora lutea * 100
postimplantation loss, for each individual pregnant animal which underwent scheduled sacrifice = (number of implantations – number of live fetuses) / number of implantations * 100
Historical control data:
- mean maternal bw during gestation, reproduction data, placenta weights, mean maternal weights, fetal external malformations/variations, fetal soft tissue malformations/variations, fetal skeletal malformations/variations
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
For details, see to table 1.
- 0/10/30/100 mg/kg bw/d dose group: 4/2/11/20 females showed reduced defecation, respectively
- no defecation was observed in 2/0/4/4 females, respectively
- exceptionally high incidence of reduced defecation in the high dose group, along with reduced food consumption indicates a treatment-related effect

For details, see to table 5.
- high-dose (100 mg/kg bw/d) group: mean food consumption distinctly and statistically significantly reduced from GD 7-23 (up to -59% in comparison to the control)
- high-dose does: 31% less food consumption compared to the concurrent control (GD 6-28)
- low- and mid-dose groups (10 and 30 mg/kg bw/d): comparable food consumption to control throughout the entire study period
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female of the control group (No.21) died after a gavage error.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
For details, see to table 3 and 4 and the attached figure
- mean body weights (BW); average body weight gain (BWC)
- high-dose (100 mg/kg bw/d) group: BW statistically significant reduced on GD 14-25 and BWC statistically significant reduced on GD 9-11
- high-dose rabbits: lost weight overall (-24.0 g vs. +104.4 g in control) during the treatment period (GD 6-28)
- no effects observed in the low- and mid-dose groups (10 and 30 mg/kg bw/d)
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
- no effects observed for the mean gravid uterus compared to the control group
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
For details, see to table 6.
spontaneous effects were observed in the control and the test group:
- watery feces or no feces in rectum: one control doe, one mid-dose doe and one high-dose doe
- rudimentary appearance of uterus and ovaries in one control doe
- granulated surface of liver in one low-dose doe
- malpositioned kidney in combination with a short ureter in one low-dose and one mid-dose doe
- absence of uterine horn(s) in one low-dose doe
- findings after gavage error (thoracic cavity filled with blood) in one control doe
Description (incidence and severity):
Two control (Nos. 7 [GD 20] and 11 [GD 28]) and four high-dose females (Nos. 78 [GD 24], 79 [GD 26], 84 [GD 28] and 94 [GD 27] - 100 mg/kg bw/d) were sacrificed after abortion ahead of schedule. Although spontaneous abortions in single does are not uncommon findings in the strain of rabbits used for this study, the high-dose cases may already represent exaggerated maternal toxicity, given also the distinct drop in food consumption and body weight gain as well as an exceptionally high number of does showing reduced defecation.

For details, see to table 2.
Number of abortions:
effects observed, treatment-related
Description (incidence and severity):
For details, see to table 2.
- abortions were observed in two control females and four high-dose females
- the females were sacrificed and excluded from calculations of mean maternal food consumption, body weight and body weight change, mean gravid uterine weights, mean organ weights, corrected (net) body weight gain and summary of reproduction data
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
For details, see to table 2.
- no test substance-related and/or biologically relevant differences between the different test groups observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
For details, see to table 7.
- no test substance-related and/or biologically relevant differences between the different test groups in the numbers of resorptions observed
Dead fetuses:
no effects observed
Description (incidence and severity):
For details, see to table 7.
- no test substance-related and/or biologically relevant differences between the different test groups in the numbers of viable fetuses
One dead fetus was found at cesarean section of one low-dose doe which may occur spontaneously in this rabbit strain.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
For details, see to table 2.
- conception rate was 92% in the control group and 96% in the low-, mid- and high-dose groups
- no test substance-related and/or biologically relevant differences between the different test groups in the conception rate observed
Other effects:
no effects observed
Description (incidence and severity):
All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age for historical control data.
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
30 mg/kg bw/day (nominal)
Basis for effect level:
body weight and weight gain
clinical signs
number of abortions
other: distinct decrease of food consumption
Dose descriptor:
NOEL
Remarks:
developmental toxicity
Effect level:
> 100 mg/kg bw/day (nominal)
Basis for effect level:
other: no test substance-related and/or biologically relevant differences for conception rate, mean numbers of corpora lutea and implantation sites or for pre- and post-implantation losses, the numbers of resorptions and viable fetuses
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
For details, see to table 8.
- high-dose (100 mg/kg bw/d) group: mean fetal weight statistically significantly lower than control in male fetuses and -12% for both sexes in comparison to the concurrent control
- mean weight of the female high-dose fetuses was also slightly lower (not statistically significanct)
- low- and mid-dose group: not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex distribution of the fetuses in all three test groups was comparable to the control fetuses. Any observable differences were without biological relevance.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- no statistically significant differences of overall incidences were noted between the groups
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- detected in single fetuses of all test groups including the control
All findings were considered to be spontaneous in origin and not treatment-related.
No statistically significant differences between the groups were noted. The overall incidences were well within the historical control range of the test facility.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- soft tissue malformations occurred in all test groups including the control
- distribution of the findings about the test groups does not indicate an association to the treatment and no statistically significant differences between the groups were noted
- total incidence of soft tissue malformations in treated animals did not differ significantly from the control group
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
VARIATIONS/UNCLASSIFIED OBSERVATIONS:

EXTERNAL (refer to table 9)
- paw hyperflexion: recorded in four fetuses of one high-dose litter, occurred together with multiple external malformations
Paw hyperflexion can be found in the historical control data at comparable incidences.
- discolored placentae: recorded in one fetus of the low-dose test group
This finding is not considered to be related to treatment.

SOFT TISSUE (refer to table 10)
- absent lung lobe (Lobus inferior medialis) in all test groups including the control observed
- for the high-dose group: not statistically significant, however, it was outside the historical control range (historical control data [HCD]: mean% 0.9, range 0.0 - 2.0); these findings were clustered in only 3 litters = litter incidence of 15% which is inside the historical control range (HCD: mean% 7.0, range 0.0 – 17.4)
Overall, this finding is a common anatomical variant in this rabbit strain. Thus, the slightly higher high-dose incidence it is not considered to be of toxicological relevance.
- other variations (e.g. cerebral ventricle, malpositioned carotid branches narrowed pulmonary trunk, dilated aorta and dilated renal pelvis) occurred in individual fetuses of low- and/or high-dose test animals as well as in control groups
The incidences of these variations were neither statistically significantly different from control nor dose-dependent and, therefore, not considered biologically relevant.
- blood coagulum around urinary bladder: in four mid-dose and three high-dose fetuses
This finding can be found in the historical control data at comparable incidences, therefore, it was neither assessed as treatment-related nor as adverse.
- empty stomach (devoid of amniotic fluid): in four fetuses of the same high-dose litter
These fetuses had multiple other findings, discussed in the section for details.

SKELETAL (refer to table 11)
- skeletal variations of different bone structures: observed in all test groups with or without effects on corresponding cartilages
The observed skeletal variations were related to several parts of fetal skeletons and appeared in the majority of cases without a relation to dosing.
- irregular ossification of interparietal: in the mid- and high-dose groups increased and outside the historical control range
This finding represents small irregularities in the shape of the ossification nuclei in the interparietal. As desmal ossification of the neurocranium continues during later development and interparietal membrane as well as surrounding bones were intact, a completely regular ossification of this bone can be expected to occur postnatally. Thus, this finding was considered to be of little, if any toxicological relevance.
- ‘unossified talus (with present cartilage)’: statistically significantly increased and outside the historical control range in the high-dose group
This finding may represent slight delays of ossification which did not affect morphology, as the underlying cartilage model was completely intact in all these cases. This assessment is supported by the fact, that the mean fetal weight of all 10 fetuses showing this finding (i.e. 18.8 g) was clearly below the mean fetal weight of all fetuses in test group 3 (33.6 g), which indicates a delay in overall development going along with the delay in ossification.
The other increased incidences of skeletal variations were either not related to dose and/or inside the historical control range. Thus, they are not considered to be associated with treatment.
The observed unclassified cartilage findings were related to the sternum and the ribs and did not show any relation to dosing. Therefore, they were assessed as not treatment-related.
Details on embryotoxic / teratogenic effects:
ASSESSMENT OF ALL OBSERVATIONS
- distribution of total malformations about the groups was not related to dose
One fetus of the control, two fetuses of the low-dose, four fetuses of the mid-dose and four fetuses of the high-dose group had more than one malformation or were multiple-malformed across the different examination areas.

For details, see table 12.
- a cluster of 4 fetuses in litter No.76 showed a spectrum of malformations and a number of less severe findings, e.g. paw hyperflexion and empty stomach (devoid of amniotic fluid)
All these findings contributed to apparently higher high-dose rates of external malformations and variations as well as unclassified soft tissue observations.
The clustered appearance in one litter and the almost identical spectrum of ontogenetically different findings in all those fetuses strongly suggests an origin of these anomalies which is unrelated to treatment. Consequently, the higher incidence of high-dose findings in their respective sections is also considered to be unrelated to treatment.

- other malformations appeared in individual fetuses of test groups 1 or 3
- no ontogenetic pattern is recognizable for the individual malformations, nor was there any cluster of any of these individual malformations
- malformations were not dose-related and all of them can be found in the historical control data at comparable or higher frequency
Overall, an association of all these findings to the treatment is not assumed.

- spontaneous origin is assumed for the external variations, soft tissue variations and the skeletal variations as observed in all test groups and the control group
If all different types of variations are summarized, none of the incidences showed a relation to dosing and can be found in the historical control data at a comparable frequency.

- spontaneous origin is assumed for the unclassified external, unclassified soft tissue and unclassified skeletal cartilage observations, observed in several fetuses of all groups
The distribution and type of these findings do not suggest any relation to treatment.
Finally, fetal examinations revealed that there is no adverse effect of the compound on the respective morphological structures up to the highest dose tested (100 mg/kg bw/d).
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Abnormalities:
no effects observed
Description (incidence and severity):
Four fetuses in one litter showed malformations and a number of less severe findings such as (among others) paw hyperflexion and empty stomach (devoid of amniotic fluid). These findings contributed to apparently higher high-dose rates of external malformations and variations as well as unclassified soft tissue observations.
The clustered appearance in one litter and the almost identical spectrum of ontogenetically different findings in all those fetuses strongly suggests an origin of these anomalies which is unrelated to treatment. Consequently, the higher incidence of high-dose findings in their respective sections is also considered to be unrelated to treatment.
Developmental effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
no
Relevant for humans:
no

Table 1: Excerpt of the results of maternal clinical signs during gestation

 

 

Day of gestation

 

 

 

Group#

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

18

19

20

21

22

23

24

25

26

27

28

TOTAL

# OF FEMALES EXAMINED

0

25

25

25

25

25

25

25

25

25

25

24

24

24

24

24

24

24

24

24

24

24

23

23

23

23

23

23

23

23

22

 

 

1

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

 

 

2

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

 

 

3

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

24

24

23

22

21

 

 

BLOOD IN BEDDING BEFORE

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

1

 

1

 

1

 

0

 

1

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

1

TREATMENT

1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

2

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

3

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

ABORTION AFTER TREATMENT

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

0

0

0

0

0

0

0

0

0

1

 

1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

2

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

3

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

0

0

0

0

0

1

ABORTION BEFORE TREATMENT

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

0

1

 

1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

2

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

3

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

1

1

0

3

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Day of gestation

 

Group#

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

18

19

20

21

22

23

24

25

26

27

28

TOTAL

STOOL/URINE

 

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

1

2

1

0

0

0

0

0

0

0

1

1

2

1

0

4

REDUCED DEFECATION BEFORE

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

1

2

1

0

0

0

0

0

0

0

1

1

2

1

0

4

TREATMENT

1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

1

0

0

0

0

0

0

0

1

1

0

0

0

0

2

 

2

0

0

0

0

0

0

0

0

0

0

0

0

0

1

2

3

2

2

2

3

3

1

0

1

2

5

3

1

3

0

11

 

3

0

0

0

0

0

0

0

0

0

1

0

0

1

4

7

17

10

9

6

9

8

8

5

6

6

5

3

5

5

0

20

REDUCED DEFECATION AFTER

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

1

2

1

0

0

0

0

0

0

0

1

1

2

1

0

4

TREATMENT

1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

1

0

0

0

0

0

0

0

1

1

0

0

0

0

2

 

2

0

0

0

0

0

0

0

0

0

0

0

0

0

1

2

3

2

2

2

3

3

1

0

1

2

5

3

1

3

0

11

 

3

0

0

0

0

0

0

0

0

0

1

0

0

1

4

7

17

10

9

6

9

8

8

5

6

6

5

3

5

5

0

20

NO DEFECATION BEFORE

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

2

0

2

TREATMENT

1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

2

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

1

1

1

1

2

3

1

0

4

 

3

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

1

3

4

3

3

3

2

2

2

1

0

0

0

0

0

4

NO DEFECATION AFTER

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

1

0

2

TREATMENT

1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

2

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

1

1

1

1

2

3

1

0

4

 

3

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

1

3

4

3

3

3

2

2

2

1

0

0

0

0

0

4

Table 2: Summary of reproduction data

 

TEST GROUP 0

0 mg/kgbw/d

TEST GROUP 1

10 mg/kgbw/d

TEST GROUP 2

30 mg/kgbw/d

TEST GROUP 3

100 mg/kgbw/d

Females Mated (N)

25

25

25

25

Pregnant (N)

23

24

24

24

Conception Rate (%)

92

96

96

96

Aborted (N)

2

0

0

4

Premature Births (N)

0

0

0

0

Dams with Viable Fetuses (N)

20

24

24

20

Dams with all Resorptions (N)

0

0

0

0

Female Mortality (N)

3 Fi

0

0

4

(%)

12

0.0

0.0

16

Pregnant at Terminal Sacrifice (N)

20 Fi

24

24

20

(%)

80

96

96

80

Corpora Lutea MEAN

9.8 D

9.8

10.8

11.0

S.D.

1.74

2.45

2.50

2.66

TOTAL

195

235

259

220

Implantation Sites MEAN

9.1 D

8.4

9.2

9.6

S.D.

2.24

2.93

2.69

3.35

TOTAL

181

201

221

192

Preimplantation Loss MEAN (%)

7.9 D

15.0

14.6

14.8

S.D.

13.88

22.83

19.55

18.32

Postimplantation Loss MEAN (%)

3.7 D

6.6

6.8

6.2

S.D.

8.47

9.64

8.66

11.52

Statistics: D = Dunnett-test (two-sided), * : p<=0.05 ** : p<=0.01; Fi =Fisher's exact test (one-sided)

Table 3: Mean maternal body weights during gestation (g)

 

 

 

TEST GROUP 0

0 mg/kg bw/d

TEST GROUP 1

10 mg/kg bw/d

TEST GROUP 2

30 mg/kg bw/d

TEST GROUP 3

100 mg/kg bw/d

DAY

0

MEAN

3755

D

3735

3738

3722

 

 

S.D.

161.8

 

174.4

165.0

163.5

 

 

N

22

 

24

24

24

DAY

2

MEAN

3820

D

3803

3813

3804

 

 

S.D.

163.3

 

174.3

159.5

157.9

 

 

N

22

 

24

24

24

DAY

4

MEAN

3869

D

3851

3847

3845

 

 

S.D.

177.5

 

176.5

167.6

156.0

 

 

N

22

 

24

24

24

DAY

6

MEAN

3932

D

3914

3908

3910

 

 

S.D.

175.9

 

192.7

170.9

157.3

 

 

N

22

 

24

24

24

DAY

9

MEAN

3969

D

3939

3944

3911

 

 

S.D.

191.9

 

186.3

194.7

179.9

 

 

N

22

 

24

24

24

DAY

11

MEAN

3982

D

3967

3965

3885

 

 

S.D.

174.7

 

191.0

191.4

196.7

 

 

N

22

 

24

24

24

DAY

14

MEAN

4036

D

4022

4020

3888*

 

 

S.D.

167.4

 

215.5

206.3

189.2

 

 

N

22

 

24

24

24

DAY

16

MEAN

4060

D

4062

4043

3875*

 

 

S.D.

167.9

 

245.2

209.3

211.7

 

 

N

22

 

24

24

24

DAY

19

MEAN

4012

D

4034

4006

3824**

 

 

S.D.

154.6

 

216.7

183.5

219.0

 

 

N

22

 

24

24

24

DAY

21

MEAN

4020

D

4006

3969

3808**

 

 

S.D.

142.0

 

233.8

185.8

228.7

 

 

N

21

 

24

24

24

 

 

 

 

 

 

 

 

DAY

23

MEAN

4026

D

4036

3994

3816**

 

 

S.D.

153.5

 

222.1

208.6

248.6

 

 

N

21

 

24

24

24

DAY

25

MEAN

4016

D

4030

4002

3848**

 

 

S.D.

163.9

 

197.0

232.9

244.3

 

 

N

21

 

24

24

23

DAY

28

MEAN

4030

D

4071

4018

3864

 

 

S.D.

202.8

 

216.6

255.6

291.3

 

 

N

21

 

24

24

20

 

 

 

 

 

 

 

 

DAY

29

MEAN

4066

D

4082

4038

3897

 

 

S.D.

179.2

 

213.0

273.2

289.9

 

 

N

20

 

24

24

20

Statistics: D=Dunnett-test (two-sided), * : p<=0.05 ** : p<=0.01

Table 4: Mean maternal body weight change during gestation (g)

 

 

 

 

 

TEST GROUP 0

0 mg/kg bw/d

TEST GROUP 1

10 mg/kg bw/d

TEST GROUP 2

30 mg/kg bw/d

TEST GROUP 3

100 mg/kg bw/d

DAYS

0

TO

2

MEAN

65.6 D

67.5

75.1

81.9

 

 

 

 

S.D.

32.00

40.53

36.07

33.40

 

 

 

 

N

22

24

24

24

DAYS

2

TO

4

MEAN

48.1 D

47.7

33.8

40.5

 

 

 

 

S.D.

36.94

36.09

36.35

33.23

 

 

 

 

N

22

24

24

24

DAYS

4

TO

6

MEAN

63.2 D

63.0

60.8

65.0

 

 

 

 

S.D.

23.22

46.02

36.95

45.39

 

 

 

 

N

22

24

24

24

DAYS

6

TO

9

MEAN

37.7 D

25.7

35.8

1.7

 

 

 

 

S.D.

56.02

79.94

67.63

68.06

 

 

 

 

N

22

24

24

24

DAYS

9

TO

11

MEAN

12.9 D

27.3

21.5

-26.6*

 

 

 

 

S.D.

53.91

27.89

50.28

48.89

 

 

 

 

N

22

24

24

24

DAYS

11

TO

14

MEAN

53.6 D

55.8

54.3

3.0

 

 

 

 

S.D.

54.88

60.38

90.43

80.20

 

 

 

 

N

22

24

24

24

DAYS

14

TO

16

MEAN

23.7 D

39.6

23.9

-13.4

 

 

 

 

S.D.

69.71

60.25

58.82

51.46

 

 

 

 

N

22

24

24

24

DAYS

16

TO

19

MEAN

-47.8 D

-28.4

-37.8

-50.1

 

 

 

 

S.D.

65.23

70.23

91.54

75.09

 

 

 

 

N

22

24

24

24

DAYS

19

TO

21

MEAN

-0.3 D

-27.5

-36.7*

-16.4

 

 

 

 

S.D.

34.96

50.66

38.55

61.69

 

 

 

 

N

21

24

24

24

DAYS

21

TO

23

MEAN

6.0 D

29.7

25.0

7.7

 

 

 

 

S.D.

40.54

56.58

62.58

67.35

 

 

 

 

N

21

24

24

24

DAYS

23

TO

25

MEAN

-10.6 D

-5.5

8.4

20.3

 

 

 

 

S.D.

73.17

57.69

68.65

72.82

 

 

 

 

N

21

24

24

23

DAYS

25

TO

28

MEAN

13.7 D

40.1

15.8

-4.2

 

 

 

 

S.D.

72.97

66.74

66.45

81.05

 

 

 

 

N

21

24

24

20

DAYS

28

TO

29

MEAN

11.4 D

11.4

19.9

32.8

 

 

 

 

S.D.

33.17

36.99

47.77

55.59

 

 

 

 

N

20

24

24

20

Statistics: D=Dunnett-test (two-sided), * : p<=0.05 ** : p<=0.01

Table 5: Mean maternal food consumption during gestation

 

 

 

TEST GROUP 0

TEST GROUP 1

TEST GROUP 2

TEST GROUP 3

 

 

 

0 mg/kg bw/d

10 mg/kg bw/d

30 mg/kg bw/d

100 mg/kg bw/d

DAYS

0 TO 6

MEAN OF MEANS

176.2

171.4

173.7

175.3

 

 

S.D.

4.60

6.55

5.07

7.45

 

 

N (days)

6

6

6

6

DAYS

6 TO 28

MEAN OF MEANS

118.2

123.0

114.1

81.6

 

 

S.D.

29.57

25.04

28.78

31.98

 

 

N (days)

22

22

22

22

DAYS

0 TO 29

MEAN OF MEANS

129.6

132.1

125.9

101.5

 

 

S.D.

35.47

30.24

35.35

47.50

 

 

N (days)

29

29

29

29

Table 6: Summary of maternal necropsy observations

 

FEMALES EXAMINED

 

N

TEST GROUP 0

0 mg/kg bw/d

25

TEST GROUP 1

10 mg/kg bw/d

25

TEST GROUP 2

30 mg/kg bw/d

25

TEST GROUP 3

100 mg/kg bw/d

25

NOTHING ABNORMAL DETECTED

N

20

22

22

20

 

%

80

88

88

80

FINDINGS AFTER GAVAGE ERROR

N

1

0

0

0

 

%

4.0

0.0

0.0

0.0

LUNGS: ACUTE FIBRINOUS -

N

1

0

0

0

PURULENT PNEUMONIA

%

4.0

0.0

0.0

0.0

LIVER: GRANULATED SURFACE

N

0

1

0

0

 

%

0.0

4.0

0.0

0.0

KIDNEY: MALPOSITIONED

N

0

1

1

0

 

%

0.0

4.0

4.0

0.0

RECTUM: NO FECES

N

1

0

2

1

 

%

4.0

0.0

8.0

4.0

WATERY FECES

N

2

0

1

1

 

%

8.0

0.0

4.0

4.0

URETER: SHORT

N

0

1

1

0

 

%

0.0

4.0

4.0

0.0

OVARIES: RUDIMENTARY

N

1

0

0

0

 

%

4.0

0.0

0.0

0.0

Table 7: Summary of reproduction data of the females

 

TEST GROUP 0

0 mg/kg bw/d

TEST GROUP 1

10 mg/kg bw/d

TEST GROUP 2

30 mg/kg bw/d

TEST GROUP 3

100 mg/kg bw/d

Pregnant at Terminal Sacrifice (N)

20

24

24

20

Resorptions: Total MEAN

0.3 D

0.5

0.7

0.6

S.D.

0.81

0.78

0.82

1.23

TOTAL

7

12

16

13

MEAN (%)

3.7 D

6.2

6.8

6.2

S.D.

8.47

9.70

8.66

11.52

Early MEAN

0.3 D

0.3

0.3

0.4

S.D.

0.80

0.69

0.48

1.14

TOTAL

6

7

8

8

MEAN (%)

3.2 D

3.8

3.6

3.7

S.D.

8.32

8.94

5.42

10.47

Late MEAN

0.1 D

0.2

0.3

0.3

S.D.

0.22

0.51

0.56

0.64

TOTAL

1

5

8

5

MEAN (%)

0.6 D

2.3

3.3

2.5

S.D.

2.48

5.72

5.80

6.52

Dead Fetuses (N)

0

1

0

0

Statistics: D=Dunnett-test (two-sided), *: p<=0.05 ** : p<=0.01

Table 8: Mean placental and fetal body weights (based on litter)

 

 

TEST GROUP 0

0 mg/kg bw/d

TEST GROUP 1

10 mg/kg bw/d

TEST GROUP 2

30 mg/kg bw/d

TEST GROUP 3

100 mg/kg bw/d

PLACENTAL WEIGHTS UNITS; GRAMS:

 

 

 

 

 

 

MEAN

5.0 D

5.1

4.8

4.7

 

S.D.

0.80

0.94

0.77

0.87

 

N

20

24

24

20

of Male Fetuses

MEAN

5.1 D

5.2

4.9

4.8

 

S.D.

0.83

0.85

0.75

1.00

 

N

20

23

23

20

of Female Fetuses

MEAN

4.9 D

5.0

4.7

4.7

 

S.D.

0.80

0.93

0.89

0.86

 

N

20

23

24

20

FETAL WEIGHTS, UNITS:

GRAMS

 

 

 

 

of all Viable Fetuses

MEAN

38.3 D

38.6

36.3

33.6*

 

S.D.

5.23

6.72

5.07

5.93

 

N

20

24

24

20

of Male Fetuses

MEAN

39.1 D

39.2

36.0

33.9*

 

S.D.

5.76

5.48

5.31

5.91

 

N

20

23

23

20

of Female Fetuses

MEAN

37.6 D

37.3

36.0

33.6

 

S.D.

5.15

6.83

5.49

6.30

 

N

20

23

24

20

Statistics: D=Dunnett-test (two-sided), * : p<=0.05 ** : p<=0.01

Table 9: Total fetal external malformations

 

 

Test group 0

0 mg/kgbw/d

Test group 1

10 mg/kg bw/d

Test group 2

30 mg/kg bw/d

Test group 3

100 mg/kg bw/d

Litter Fetuses

N N

20

174

24

189

24

205

20

179

Fetal incidence

 

N (%)

 

1 (0.6)

 

1 (0.5)

 

0.0

 

5 (2.8)

Litter incidence

 

N (%)

 

1 (5.0)

 

1 (4.2)

 

0.0

 

2 (10)

Affected fetuses/litter

 

Mean %

 

0.5

 

0.4

 

0.0

 

2.4

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 10: Total soft tissue malformations

 

 

Test group 0

0 mg/kgbw/d

Test group 1

10 mg/kg bw/d

Test group 2

30 mg/kg bw/d

Test group 3

100 mg/kg bw/d

Litter Fetuses

N N

20

174

24

189

24

205

20

179

Fetal incidence

 

N (%)

 

2 (1.1)

 

3 (1.6)

 

3 (1.5)

 

1 (0.6)

Litter incidence

 

N (%)

 

1 (5.0)

 

3 (13)

 

2 (8.3)

 

1 (5.0)

Affected fetuses/litter

 

Mean %

 

0.9

 

1.6

 

1.4

 

0.4

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 11: Total skeletal malformations

 

 

Test group 0

0 mg/kgbw/d

Test group 1

10 mg/kg bw/d

Test group 2

30 mg/kg bw/d

Test group 3

100 mg/kg bw/d

Litter Fetuses

N N

20

174

24

189

24

205

20

179

Fetal incidence

 

N (%)

 

1 (0.6)

 

2 (1.1)

 

2 (1.0)

 

1 (0.6)

Litter incidence

 

N (%)

 

1 (5.0)

 

2 (8.3)

 

2 (8.3)

 

1 (5.0)

Affected fetuses/litter

 

Mean%

 

0.6

 

1.2

 

1.1

 

0.4

Table 12: Fetuses with more than one malformation

Test group

Doe No.-Fetus No., Sex

Finding

0 (0 mg/kg bw/d)

25-10 F

domed head, hydrocephaly

1 (10 mg/kg bw/d)

26-10 M

thoracic hemivertebra, misshapen thoracic vertebra

44-01 F

malpositioned kidney, short ureter

2 (30 mg/kg bw/d)

68-07 F

exoccipital fused with 1st cervical arch, cervical hemivertebra

69-03 M

multiple malformations of the great vessels (persistent truncus arteriosus, aortic arch atresia, malpositioned subclavian origin)

75-02 F

aortic arch atresia, malpositioned kidney

75-06 F

thoracic hemivertebra, branched rib

3 (100 mg/kg bw/d)

76-04 M

multiple external malformations (domed head, cleft palate, small tongue), hydrocephaly

76-06 F

multiple external malformations (domed head, cleft

palate, small tongue)

76-11 F

multiple external malformations (domed head, cleft

palate, small tongue), severely malformed skull bones

76-12 M

multiple external malformations (domed head, cleft

palate, small tongue)

mg/kg bw/d = milligram per kilogram body weight per day; No.= number; M = male; F = female

The stability of the test substance in 0.5% CMC suspension in deionized water (with 10 mg/100 mL Cremophor EL) over a maximum of 7 days and warmed up to approximately 30 degrees Celsius was demonstrated. The homogeneous distribution of the test substance in the vehicle was shown. The correctness of the prepared concentrations was shown.

Conclusions:
The oral administration of the test substance to pregnant New Zealand White rabbits from implantation to one day prior to the expected day of parturition (GD 6-28) caused evidence of systemic maternal toxicity at the high-dose level of 100 mg/kg bw/d, such as a slightly higher incidence of abortions and reduced defecation in almost all females of this group, along with a distinct decrease of food consumption as well as body weight/body weight gain.
In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 30 mg/kg bw/d.
The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is also set at 30 mg/kg bw/d. The mean fetal body weights were significantly lower in the 100 mg/kg bw/d group compared to the control group (33.6 g versus 38.6 g). Considering the immediate effect of dosing on maternal food consumption and body weight loss during GD 6-28, the reduced fetal body weights are a result of the poor nutritional condition of the does. The substance has no direct developmental toxicity. No teratogenicity was observed.
Effect on developmental toxicity: via oral route
Dose descriptor:
NOAEL
18 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Information on developmental toxicity from is available from an OECD 422 screening study, an OECD 421 'plus' dose range finding study, as well as an OECD 443 extended one-generation study.


For detailed study information please refer to additional information in the chapter effect on fertility.

Justification for classification or non-classification

The available experimental data does meet the criteria laid out for classification and labelling in EC Regulation 1272/2008. As a result the substance is not considered to be classified for toxicity to reproduction under Regulation (EC) No. 1272/2008, as amended for the second time in Directive (EC 286/2011).


Overall, lower numbers of implants with subsequently smaller litter sizes were likely caused by considerable stress through maternal toxicity at 1800 ppm, which was particularly distinct during gestation/lactation. Furthermore, no changes were seen in estrous cycle data, sperm quality, mating behavior, conception, gestation, parturition, lactation, and weaning as well as sexual organ weights and morphology.


Regarding developmental toxicity, lower pup body weights were attributed to direct exposure of the offspring to the test compound, resulting in a general delay of development. Furthermore, pup body weight effects had no influence on postnatal pup survival or well-being. Intermediate and high dose F1 and F2 pups revealed no adverse findings during early lactation or later, in clinical and/or gross necropsy examinations. Significant delays in vaginal opening and preputial separation in F1 offspring animals (which were however within HCD) were regarded as most likely a consequence of systemic toxicity and subsequent general developmental delay.


Histopathological findings in thyroids and thyroid hormone changes were seen as a consequence of liver cell enzyme induction and no direct effect of the test substance on the hormonal system.


Regarding findings in the spinal cord of cohort 2A animals, further tests have been scheduled to be able to make a final conclusion wether the findings in neurohistopathology should be regarded as developmental effects or rather as  chronic toxicity effects.


For more details refer to the part additional information.

Additional information