Benzenamine, N-phenyl-, reaction products with 2,4,4-trimethylpentene

Toxicity to reproduction

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Benzenamine, N-phenyl-, reaction products with 2,4,4-trimethylpentene

EC Number:

270-128-1

EC Name:

Benzenamine, N-phenyl-, reaction products with 2,4,4-trimethylpentene

Cas Number:

68411-46-1

Molecular formula:

C16H19N - C28H43N (main constituents)

IUPAC Name:

Benzenamine, N-phenyl-, reaction products with 2,4,4-trimethylpentene

Test material form:

liquid: viscous

Details on test material:

Chemical Identity: Benzenamine, N-phenyl-, reaction products with 2,4,4- trimethylpentene
Batch 50116118D
Expiry date: 25 Dec 2020
Physical state/Appearance: Viscous liquid / clear/ light orange
Storage conditions: Room temperature

Specific details on test material used for the study:

- Content: 100g/100 g UVCB

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Remarks:

Control group

Doses / concentrations 2

Dose / conc.:

200 ppm

Remarks:

Low dose group; Approx. 18 mg/kg bw/d

Doses / concentrations 3

Dose / conc.:

600 ppm

Remarks:

Intermediate dose group; Approx. 54 mg/kg bw/d

Doses / concentrations 4

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

Examinations

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 (%)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

54 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: 600 ppm

2

Dose descriptor:

LOAEL

Remarks:

systemic toxicity

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: 200 ppm

Results: P1 (second parental generation)

General toxicity (P1)

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 / performance (P1)

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.

Effect levels (P1)

1

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

54 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: 600 ppm

2

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

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: 200 ppm

Results: F1 generation

General toxicity (F1)

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

Developmental neurotoxicity (F1)

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.

Effect levels (F1)

1

Dose descriptor:

NOAEL

Remarks:

developmental neurotoxicity

Generation:

F1 (cohort 2A)

Effect level:

167 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

1800 ppm

2

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

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: 200 ppm

Results: F2 generation

General toxicity (F2)

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.

Effect levels (F2)

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

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: 200 ppm

Overall reproductive toxicity

Reproductive effects observed:

yes

Lowest effective dose / conc.:

1 800 ppm

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Applicant's summary and conclusion

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. However, the same dose (200 ppm) was determined the NOAEL in the F1 adult rats. 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 smaller litters at the dose 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 pre-weaning body weight gain, which was observed at the dose level of 600 ppm (about 54 mg/kg bw/d). Pup mortality was unchanged and no malformations were observed in the progeny at any of the doses tested. An increased incidence of focal to multifocal axonal degeneration in the white matter of thoracic cord was observed at PND 77 at the dose level of 1800 ppm, however this finding could not be confirmed in pups at PND 22. Therefore, this finding was considered a chronic rather than developmental toxicity finding. In conclusion, no developmental neurotoxicity was observed under the conditions of the study.