N-methoxy-1-(2,4,6-trichlorophenyl)propan-2-amine

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Remarks:

The two-generation reproductive toxicity study was conducted solely to comply with non-EU national registration requirement, and has been provided here in accordance with REACH, Article 22(1)e.

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 November 2015 to 06 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

2001

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Version / remarks:

1998

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: JMAFF, 12 - Nousan No. 8147

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

methoxy[1-(2,4,6-trichlorophenyl)propan-2-yl]amine

EC Number:

941-634-6

Cas Number:

1228284-78-3

Molecular formula:

C10H12Cl3NO

IUPAC Name:

methoxy[1-(2,4,6-trichlorophenyl)propan-2-yl]amine

Test material form:

other: liquid

Details on test material:

- Physical state: Brown liquid
- Expiration date of the lot/batch: Recertification date end May 2017
- Storage condition of test material: Room temperature (<30°C), protected from light and humidity

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat is a suitable rodent species, acceptable to regulatory authorities and for which extensive background data are available. The Han Wistar rat is commonly used in reproduction studies because of the good fertility and fecundity of the strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, CT9 4LT, England
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: The males were 5 to 6 weeks of age and the females were 4 to 5 weeks of age on arrival (P); From Day 20 of age (F1)
- Weight at study initiation: (P) Males: 182 g to 266 g; Females: 123 g to 169 g.; (F1) Males: x-x g; Females: x-x g
- Fasting period before study: No
- Housing: The P generation animals and selected F1 generation animals were housed in groups of 4, by sex, in grid-floor cages suspended over paper-lined trays, until pairing and for males post-pairing. For pairing, 1 male and 1 female from the same group (avoiding sibling mating for F1 animals) were housed together in grid-floor cages suspended over paper-lined trays. On confirmation of mating, the males were returned to the group cages and the mated females were housed individually and subsequently with their litter, in solid-floor cages with appropriate bedding material. Animals were also housed individually on welfare grounds, where required.
- Diet and water (e.g. ad libitum): A pelleted rodent diet, VRF1 (manufactured by SDS) supplied by Charles River (UK) Limited, Margate, Kent, CT9 4LT, England, and mains tap water (in bottles) were freely available.
- Acclimation period: 11 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 °C to 23 °C
- Humidity (%): 40 % to 70 %
- Photoperiod (hrs dark / hrs light): fluorescent light set to give a cycle of 12 hours light and 12 hours dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: suspension in 1 % (w/v) carboxymethylcellulose with 1 % (v/v) Tween 80

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated within the known stability period, for each group separately, as a suspension in 1 % (w/v) carboxymethylcellulose with 1 % (v/v) Tween 80. The purity of the test item was not accounted for in the dose calculations.

The test item was checked visually to ensure homogeneity before preparation. A weighed quantity of test item was added to a container and approximately 90 % of the final weight of vehicle was added gradually, whilst stirring. The formulation was then stirred and heated to a maximum of 50 °C until a homogeneous mixture was formed. The formulation was made up to final weight with vehicle and then divided between amber glass bottles for dosing and stored refrigerated (2 °C to 8 °C).

Formulations were removed from refrigerated storage and stirred for at least 15 minutes before the start and until completion of dosing, to ensure thorough re-suspension and homogeneity.

Details on mating procedure:

After the pre-pairing dosing period (approximately 10 weeks), each female was paired with a male from the same dose group for up to 14 days. P generation Male 68 (10 mg/kg/day) died on Day 2 of the pairing period; this male had been paired for mating with Female 164. This female was re-paired with a male from the same dose group, which had mated previously, from Day 3 of the pairing period onwards (Male 54).

On confirmation of mating, the males were returned to the group cages and the females were housed individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were taken from each formulation. The test item formulations prepared for use on the first day of dosing were analysed for the test item using a validated method to confirm homogeneity and achieved concentration. To aid further assessments, test item formulations prepared for use during Weeks 0/1 and Weeks 1/2 were also analysed to assess homogeneity and achieved concentrations. Having confirmed homogeneity for these formulations, samples were then taken from all test item formulations prepared every 2 months thereafter and analysed to confirm concentration only. For formulations prepared for Control animals, samples taken from formulations prepared for the first day of dosing, for use during Week 0/1 and for use every 2 months thereafter (from the time the test item formulations were confirmed as homogeneous) were analysed to confirm absence of test item.

All remaining samples were retained and will be discarded within 1 month of finalisation of this report.

Duration of treatment / exposure:

At least 10 weeks before pairing and then until the day before necropsy. Males were dosed before, during and after pairing for mating and females were dosed before and during pairing, during gestation and until Day 20 of lactation. Animals that were mid-parturition at the time of dosing were not dosed on that day. The selected F1 generation animals were dosed from Day 21 of age; an exception to this was for Female 393 which was dosed from Day 23 of age after replacing Female 369. Individual dose volumes were based on the most recently recorded body weight using a dose volume of 5 mL/kg/body weight.

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2.5 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

10 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

50 mg/kg bw/day

No. of animals per sex per dose:

24 males and 24 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were selected on the basis of results from a 90-day oral (gavage) toxicity study in the rat (OECD 408, Sequani Study Number: BFI0318) and an oral (gavage) reproduction /developmental toxicity screening test (OECD 421, Sequani Study Number: BFI0319). The top dose level of 75 mg/kg/day in these studies was considered to be in excess of the maximum tolerated dose (MTD) based on adverse effects on body weight gain and excessive liver toxicity; therefore, a top dose level of 50 mg/kg/day was selected for this study as it was expected to represent the MTD for rats in a study of this type. The low dose, 2.5 mg/kg/day, was expected to represent a clear NOAEL, and the mid-dose level of 10 mg/kg/day was selected to provide information on the dose response relationship of test item-related effects in the target organs for toxicity.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were examined twice daily for mortality and morbidity. From the start of dosing, all animals were examined daily for clinical signs of toxicity or changes in behaviour and appearance


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: each animal was given a detailed clinical examination once each week.

BODY WEIGHT: Yes
- Time schedule for examinations: Male body weights were recorded on the first day of dosing, at weekly intervals throughout the study and then on the day of necropsy. Female body weights were recorded on the first day of dosing and then at weekly intervals until the day of mating. Females were also weighed on Days 0, 7, 14 and 20 of gestation and on Days 0 (where required for dose volume calculations), 1, 4, 7, 14 and 21 of lactation and on the day of necropsy. Females where all the pups had died in the litter before Day 21 of age, were weighed on Days 1, 4, 7, 14 and 21 of lactation.

Females that failed to litter were weighed weekly until necropsy to calculate the dose volume to be administered; these data have not been reported.

FOOD INTAKE:
The amount of food consumed by each cage of males was recorded at weekly intervals during their pre-pairing and post-pairing periods.
Food intake of the females was recorded weekly during the pre-pairing period and over Days 0 to 4, 4 to 7, 7 to 10, 10 to 14, 14 to 17 and 17 to 20 of gestation and over Days 1 to 4, 4 to 7, 7 to 10, 10 to 14, 14 to 17 and 17 to 21 of lactation.
Food intake of females where all pups died in the litter before Day 21 of age were recorded on Days 1 to 4, 4 to 7, 7 to 10, 10 to 14, 14 to 17 and 17 to 21 of lactation.
Food intake was not recorded for females that failed to mate or litter.

Oestrous cyclicity (parental animals):

For 21 days before the start of the pairing period, vaginal smears were taken daily by lavage. The smear was examined under light microscopy and the stage of the oestrous cycle was determined by the type of cell present.

Sperm parameters (parental animals):

Sperm motility and concentration were assessed for all males killed at scheduled necropsy using the Hamilton Thorn IVOS Computer Assisted Sperm Analysis (CASA) system. The assessment was performed using fluid from one cauda epididymis. The cauda epididymis was weighed separately.

A sample of the epididymal fluid was retained in neutral buffered formalin, a smear was prepared for each Control and high dose male and at least 200 sperm per sample were examined for morphological abnormalities.

After weighing of the testes, the tunica albuginea of one testis was removed and the testis was snap frozen in liquid nitrogen and stored at -20 °C until required. For each Control and high dose male, the testis was thawed, homogenised and the resistant spermatids counted using the CASA system.

Litter observations:

The females were allowed to rear their offspring to weaning on Day 21 of lactation. Abnormalities of nesting or nursing behaviour were recorded.
for F1a and F2a animals, The total litter size was recorded after completion of parturition and daily thereafter. Numbers of each sex were recorded daily from Day 1 of age. On Day 4 of age, the size of each litter was adjusted by eliminating extra pups to yield, as nearly as possible, 4 males and 4 females. Litters of fewer than 8 pups were not altered. Pups were selected on a total randomisation basis. Non-selected pups were killed and discarded using a suitable Schedule 1 method.

Day 0 of age for the pups (equivalent to Day 0 of lactation for observations assigned to the dams) was defined as the day of completion of littering.

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The total litter size was recorded after completion of parturition and daily thereafter. Numbers of each sex were recorded daily from Day 1 of age. On Day 4 of age, the size of each litter was adjusted by eliminating extra pups to yield, as nearly as possible, 4 males and 4 females. Litters of fewer than 8 pups were not altered. Pups were selected on a total randomisation basis. Non-selected pups were killed and discarded using a suitable method.
Day 0 of age for the pups (equivalent to Day 0 of lactation for observations assigned to the dams) was defined as the day of completion of littering.

Animals were examined twice daily for mortality and morbidity. All pups were examined daily for malformations and clinical signs of toxicity.


The following parameters were examined in [F1] offspring:
Quantitative evaluation of follicles was performed for the F1 generation only. For all F1 females, the centre of the left ovary was sectioned using the butterfly technique (5 sections, approximately 100 µm apart) and stained with haematoxylin and eosin for microscopic examination. The following follicles were then counted for Control and high dose females:
• Primordial follicles and naked oocytes (Types 1 and 2 on the Pederson and Peters classification).
• Primary follicles (Type 3a on the Pederson and Peters classification).

The anogenital distance of the F2a animals was measured on Day 1 of age.

Pups were weighed individually on Days 1, 4, 7, 14 and 21 of age.

Particular attention should be paid to the external reproductive genitals which should be examined for signs of altered development; gross evaluation of external genitalia]

GROSS EXAMINATION OF DEAD PUPS:
[no / yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead]

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: The males were killed approximately 6 weeks after completion of the mating phase (after successful littering). The animals were killed by exposure to carbon dioxide gas in a rising concentration.
- Maternal animals: Females with litters were killed on Day 21 of lactation at weaning of their litters. The remaining females, including those apparently non-pregnant and those where all the litter had died before Day 21 of age, were also killed at this time.

GROSS NECROPSY
A dead body weight was recorded and the cranial, thoracic and abdominal cavities were opened by a ventral mid-line incision and the major organs and uterus were examined. The number of implantation scars/sites for each female was recorded. The uterus of any apparently non-pregnant female was given a visual assessment under magnification to confirm pregnancy status. Organs or tissues showing any macroscopic abnormalities were recorded and retained. For each male, one epididymis was processed for sperm evaluation.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs were weighed after trimming of fat and other contiguous tissue (contralateral organs were weighed together):

adrenals, pituitary, brain, prostate, epididymides, seminal vesicles (including coagulating gland), kidneys, spleen, liver, testes, ovaries, thyroids (including parathyroids), uterus (including uterine cervix and oviducts)

For all animals, with the exception of the testes, either whole organs or samples of the tissues listed below, were preserved in neutral buffered formaldehyde. The testes were fixed in modified Davidson’s solution.

adrenal glands, prostate & seminal vesicles (including coagulating gland), brain, epididymides, spleen, Kidneys, testes, liver, thyroids (including parathyroids), ovaries, uterus (including uterine cervix and oviducts), pituitary, vagina

For all animals, the tissues specified in the tissue list were wax embedded, cut at a nominal thickness of 4 µm to 5 µm and stained with haematoxylin and eosin. For all Control and high dose animals, any premature decedents and any non-pregnant females or females that had lost their litter, the tissues specified in the tissue list were examined microscopically.

Additionally, reproductive organs of the low and intermediate dose animals suspected of reduced fertility (i.e. males and females that failed to mate, any males that failed to sire a pregnancy and any non-pregnant females or females that failed to deliver healthy offspring) were also examined microscopically.

As macroscopic abnormalities of the kidneys were noted at necropsy, the kidneys of all F1 generation males were wax embedded, cut at a nominal thickness of 4 µm to 5 µm and stained with haematoxylin and eosin. Only the kidneys from the Control and high dose group were examined in the first instance, with tissues from the low and intermediate dose groups examined after findings were identified in the high dose group.

Following microscopic examination and external peer review, microscopic changes were detected in the liver of the high dose males and females (P and F1 generations). Consequently, the livers from the low and intermediate dose groups were examined microscopically.

Postmortem examinations (offspring):

SACRIFICE
- With the exception of pups culled on Day 4 of lactation, which were not examined further, a necropsy was conducted on all pups killed or found dead during lactation and all unselected pups from Day 21 of age. The pups were killed by an intraperitoneal injection of sodium pentobarbitone solution (for those up to the age of 14 days) or by exposure to carbon dioxide gas in a rising concentration (for older pups). Where possible, a dead body weight was recorded for pups where organ weights were to be determined. The cranial (where required), thoracic and abdominal cavities were opened by a ventral mid line incision and the major organs were examined.
F1a and F2a animals were identified at necropsy; numbers were allocated as the dam number followed by a two digit pup identifier

- These animals were subjected to postmortem examinations (macroscopic examination) as follows:
For 1 male and 1 female pup per litter, the tissues listed below were retained. All other pups had a gross macroscopic examination and only gross abnormalities were retained.
For all animals, either whole organs or samples of the tissues listed below were preserved in neutral buffered formaldehyde.
brain, spleen, thymus, all gross lesions

Due to the age and size of early decedent pups, and to prevent damage to any tissues during removal, the affected tissues were left in situ and the whole carcass was retained in neutral buffered formaldehyde.


ORGAN WEIGTHS
For 1 male and 1 female pup from each litter, the following organs were weighed after trimming of fat and other contiguous tissue:
brain, spleen, thymus

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 50 mg/kg/day CA5204A, excessive salivation was observed at the time of dosing for most of the dosing period.

There were no other clinical observations considered to be related to the test item.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two early decedents were attributed to an intubation error from dosing. One male given 10 mg/kg/day was found dead in the cage during the pairing period (Male 68) and 1 female given 50 mg/kg/day was euthanised following a decline in clinical condition during the pre-pairing period (Female 177). For the male, decreased activity and slow and laboured breathing were noted the day before death and the macroscopic necropsy identified a rupture to the oesophagus. Microscopic examination confirmed the perforation in the oesophagus and revealed pericarditis, pleurisy and abscessation with foreign material in the thoracic cavity. For the female, clinical signs included decreased activity, slow and laboured breathing, abnormal and unsteady gait, hunched posture, piloerection and partially closed eyes. Whilst a rupture was not identified at necropsy, macroscopically there was a thickened and discoloured region in the oesophagus and microscopic examination revealed severe oesophageal ulceration. One female given 10 mg/kg/day was also euthanised due to difficulties in parturition on Day 22 of gestation (Female 148). This isolated incidence of dystocia was considered not to be related to the test item.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 50 mg/kg/day, the males gained slightly less weight than the Controls from Week 4 onwards, so that the group mean body weight was approximately 6 % lower than Controls by the end of the P generation; this difference from Controls was not statistically significant.

There was no adverse effect on body weight for the females given 50 mg/kg/day or on the males and females given 2.5 or 10 mg/kg/day. During lactation, the females from all groups given the test item gained slightly more weight than Controls; this was considered not to be toxicologically adverse.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

For the males, there was no effect of the test item on food intake.

Females given 50 mg/kg/day ate slightly less than the Controls during lactation (p≤0.05 from Day 10 of lactation onwards); however, since the reduction was only minor and did not result in reduced body weights, this was considered not to be toxicologically adverse. There were no effects on food intake during the pre-pairing period or gestation for females given 50 mg/kg/day, or for females given 2.5 or 10 mg/kg/day throughout the dosing period.

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Findings considered to be related to the test item were seen in the liver. There was centrilobular hepatocyte hypertrophy in males from all groups given the test item and in females given 10 or 50 mg/kg/day. In addition, centrilobular hepatocyte vacuolation was found in males given 10 or 50 mg/kg/day and in a single female given 50 mg/kg/day. The incidence/severity of these changes were dose-related.
Although hepatocyte hypertrophy was observed at all dose levels, there was no corresponding increase in liver weight for the groups given 2.5 or 10 mg/kg/day. Whilst an increase in liver weight is often associated with this change, the incidence of this finding, particularly for the males, was considered to demonstrate an response to test item administration.

There were no other microscopic changes associated with the test item.

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There was no effect of the test item on either the mean number of oestrous cycles or the mean cycle length.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There was no adverse effect of the test item on sperm motility or concentration at any dose level.

Sperm morphology and homogenisation resistant testicular spermatid counts in the group given 50 mg/kg/day were similar to the Controls.

Reproductive performance:

no effects observed

Description (incidence and severity):

There was no effect of the test item on fertility or mating performance at any dose level.
There was no overall effect of the test item on the mean duration of gestation or parturition.
There was no effect of the test item on the mean number of pups born alive.

Postnatal survival was affected at 50 mg/kg/day only. Although the number of live pups up to Day 7 of age for the group was comparable with Controls, from Day 7 of age onwards there were sporadic pup deaths, most prominent in a small number of the litters, with total litter loss for 1 female only (Female 184). These deaths resulted in a slight reduction in the survival index for the overall lactation period, which did not achieve statistical significance. There was no clear effect on pup survival in the groups given 2.5 or 10 mg/kg/day.

Total litter loss was noted for 1 Control group female (Female 111); this female gave birth to 1 pup only.

There was no effect on the mean pup sex ratio.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Target system / organ toxicity (P0)

Critical effects observed:

yes

Lowest effective dose / conc.:

10 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 50 mg/kg/day, excessive salivation was observed at the time of dosing for most of the dosing period.

There were no other clinical observations considered to be related to the test item.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There no deaths associated with the test item.

Two females were killed following a decline in clinical condition, as a result of an intubation error. One female from the 50 mg/kg/day group was euthanised at the start of the F1 generation (Female 369 - replaced by Female 393) and 1 female given 10 mg/kg/day was killed during the lactation period (Female 352); consequently all of the pups in this litter were also killed. In both instances microscopic pathology confirmed the decline in clinical condition was a result of an intubation error.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no adverse effect of the test item administration on body weight gain. Animals given 50 mg/kg/day were slightly lighter than Controls at the start of the F1 generation (p≤0.05 for the males), however, the animals gained weight so that by Week 2 of this study phase, body weights were generally comparable across the groups.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At 50 mg/kg/day, mean absolute, adjusted and relative to body weight liver weights were higher than Controls (27.4 %, 30.1 %, 29.6 % respectively for the males and 24.1 %, 22.5 %, 21.2 % respectively for the females (p≤0.01)); this was considered to correlate with the microscopic changes in the liver. Seminal vesicle and thyroid weights (absolute, adjusted and body weight related) were also higher than Controls for males given this dose; the data attained statistical significance for the adjusted seminal vesicle weight (p≤0.05) and for absolute and adjusted thyroid weights (p≤0.01). For the seminal vesicles, there were no correlating microscopic changes and although full microscopic examination was not conducted for thyroids, the individual body weight-related weights for this organ were typically within the historical control data ranges (see Appendix 19). On this basis, the apparent effect on the weight of these organs was considered not to be adverse.

Kidney weights were also higher than Controls for males given 10 or 50 mg/kg/day, with adjusted values attaining statistical significance (p≤0.05 and p≤0.01, respectively).

At 2.5 mg/kg/day, adrenal weights for the males (absolute, adjusted and body weight related) were lower than Controls. In the absence of a dose response, this was considered a result of individual variation and not an effect of the test item.

There were no effects on organ weights for females given 2.5 or 10 mg/kg/day.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

For males given 10 or 50 mg/kg/day, a mottled discolouration to the kidneys was reported; this was noted for 1 male given 10 mg/kg/day and 8 males in the group given 50 mg/kg/day. Although there were microscopic findings reported in the kidneys of animals in these groups, it is considered that the macroscopic and microscopic findings were not related.

There were no test item-related macroscopic abnormalities for the females at all dose levels, or for the males given 2.5 mg/kg/day.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic findings associated with the test item were noted in the liver. Centrilobular hepatocyte hypertrophy and/or centrilobular hepatocyte vacuolation were found in males given 10 or 50 mg/kg/day and in females given 50 mg/kg/day. The incidence/severity of these changes were dose-related.
In the kidneys, an increased incidence of minimal basophilia in the cortical tubules was seen in the males given 50 mg/kg/day when compared with Controls. Since the finding was still graded as minimal in all affected males and this is a relatively common finding, it was considered not to be a clear evidence of toxicity.

The spectrum of all other microscopic findings was consistent with changes encountered in rats of this age kept under laboratory conditions.

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There was no effect of the test item on either the mean number of oestrous cycles or the mean length of each cycle.

One female given 2.5 mg/kg/day did not cycle before the start of the pairing period (Female 325). In the absence of similar findings at higher dose levels, this isolated instance was not associated with the test item.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Sperm motility and concentration were unaffected by the test item administration.

Morphology and homogenisation resistant testicular spermatid counts were comparable for the Controls and group given 50 mg/kg/day.

Reproductive performance:

no effects observed

Description (incidence and severity):

There was no effect of the test item on the fertility or mating performance of the F1 generation. With the exception of 1, 1 and 2 females in the Control and groups given 2.5 or 10 mg/kg/day, respectively, the females typically mated within 4 days of pairing. For the females which failed to mate, this coincided with the absence of oestrous cycling during the pairing period, despite the fact that most animals cycled before pairing.
Of the mated females, only 2 animals in the high dose group were not pregnant. Since the remaining 22 females given this dose were pregnant, this was considered to be unrelated to test item administration.
The length of gestation was similar across the groups, with all pregnant females giving birth to live offspring.
There was no effect of test item administration on the mean number of live pups born alive or on the percentage of male pups. In contrast to the P generation, the postnatal development of the offspring was also unaffected, with survival indices comparable across the groups.

Effect levels (P1)

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Target system / organ toxicity (P1)

Critical effects observed:

yes

Lowest effective dose / conc.:

50 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were slow and/or laboured breathing associated with the test item administration at 50 mg/kg/day.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

At 50 mg/kg/day, there was an increased number of pup deaths from Day 7 onwards, compared with Controls. The pups were typically either found dead or euthanised due to a decline in clinical condition (including signs of slow and/or laboured breathing, skin tenting and no milk in stomach). At necropsy, the most prominent macroscopic abnormality in the pups was the absence of milk in the stomach.
Pup deaths were also observed in the groups given 2.5 or 10 mg/kg/day, however, since the incidences were similar to those observed in the Controls, these deaths were considered not to be associated with test item.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

For all groups given the test item, the pups were marginally lighter than Controls after birth, achieving statistical significance at 50 mg/kg/day only (p≤0.05). Whilst the pups gained weight thereafter, at 50 mg/kg/day the F1a animals remained significantly lighter than Controls throughout lactation (p≤0.01) and they gained less weight (p≤0.05). Pups from the groups given 2.5 or 10 mg/kg/day gained weight so that the group mean body weight was similar to the Controls by the end of lactation.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

effects observed, non-treatment-related

Description (incidence and severity):

At 50 mg/kg/day, the males were approximately 2 days older than the Controls when balanopreputial separation was complete (p≤0.05) and the group mean body weight was also slightly greater on the day of sexual maturation; however, the age of attainment for the individuals was within the individual data for the Controls from this study and the historical control ranges, with the maximum individual body weight on attainment only marginally higher. The females given this dose were unaffected, with the day of vaginal opening comparable with the Controls.

There was no effect on sexual development for the groups given 2.5 or 10 mg/kg/day.

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

At 50 mg/kg/day, the absolute and adjusted brain weights for the males and females were marginally lower than the Controls; the difference was statistically significant for the absolute weights for the males and females (p≤0.01) and for the adjusted weights for females (p≤0.05). This difference was considered to reflect the lower body weight for the animals, rather than a direct effect on brain weight, as brain weights as a percentage of body weight were comparable with Controls.
Also at 50 mg/kg/day, absolute spleen and thymus weights were slightly lower than Controls for the females only (p≤0.05); however, adjusted spleen and thymus weights were not significantly different to Controls, and so the difference therefore, was considered to reflect the lower body weight for these animals.

There were no other differences in organ weights for the F1a pups at 2.5 or 10 mg/kg/day.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related macroscopic necropsy findings in the F1a animals.

Histopathological findings:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1a

Effect level:

10 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

Target system / organ toxicity (F1)

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

Clinical signs:

no effects observed

Description (incidence and severity):

There wereclinical signs for the pups that were associated with test item administration.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no deaths or clinical signs for the pups that were associated with CA5204A administration.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no effect of the test item on absolute pup body weight or pup body weight gain; data were comparable across the groups.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

Anogenital distance was considered to have been unaffected by CA5204A.

The mean anogenital distance adjusted for body weight (cubed root) for female pups from the test item groups was slightly longer than the Controls, with the result attaining statistical significance (p≤0.01). Since there was a large degree of variation in all groups, the values for individuals were comparable between the Control and the groups given the substance, and the mean values were comparable with the historical control range (Appendix 18), this finding was considered not to be associated with the test item.

There was no notable difference in the mean anogenital distance for the male pups across the groups.

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no effects on mean brain, spleen or thymus weights for the F2a animals that were associated with the test item.

At 2.5 mg/kg/day, the mean spleen weight was higher than the Controls, with the difference for the male pups attaining statistical significance (p≤0.05). In the absence of a similar increase at higher dose levels, this was considered not to be associated with the test item.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related macroscopic findings in the F2a animals.

Histopathological findings:

not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

Dose descriptor:

NOEL

Generation:

F2a

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

gross pathology

Target system / organ toxicity (F2)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Administration of the substance, once daily, by oral gavage at 2.5, 10 or 50 mg/kg/day to male and female Crl:WI(Han) rats for two successive generations was generally well tolerated, with no effect on reproductive performance, mating behaviour or conception. In terms of systemic toxicity, effects were primarily limited to animals given 50 mg/kg/day.Therefore, the No-Observed-Adverse-EffectLevel (NOAEL) for effects on reproduction was considered to be 50 mg/kg/day and the No Observed-Adverse-Effect-Level (NOAEL) for systemic toxicity was considered to be 10 mg/kg/day.

Executive summary:

Four groups of 24 male and 24 female rats of the Crl:WI(Han) strain were dosed by oral gavage at dose levels of 0 (Vehicle), 2.5, 10 or 50 mg/kg/day N-methoxy-1 -(2,4,6 -trichlorophenyl)propan-2 -amine for 10 weeks before pairing, during pairing, gestation and lactation, and until necropsy.  Four groups of 24 males and at least 24 females were selected from the weaned parental (P) generation litters to form the filial (F1) generation; these animals were dosed once daily by oral gavage at the same dose levels as the P generation from Day 21 of age for approximately 10 weeks before pairing, during pairing, gestation and lactation and until necropsy.  Animals were dosed using a dose volume of 5 mL/kg.  

All animals were examined for effects on general condition, body weight and food intake.  The stage of the oestrous cycle was recorded for 21 days before pairing for P and F1 parental females, and during the pairing period, vaginal smears were taken daily until sperm were found in the smear.  The females were allowed to litter and rear their offspring to weaning.  The day of sexual development was recorded for all selected F1 generation animals.

The P and F1 parental males were subjected to macroscopic necropsy once successful littering was completed.  The testes and epididymides were removed and weighed and sperm evaluation was conducted.  The P and F1 generation parental females were killed and subjected to necropsy on Day 21 of lactation.  A macroscopic necropsy was performed and the number of implantation scars was recorded.  For the P and F1 parental males and females, a selection of organs were weighed, fixed and examined microscopically.  Unselected P generation pups (F1a) and all F1 generation pups (F2a) were killed on, or shortly after, Day 21 of age.  A gross macroscopic necropsy was performed on all pups and, for one male and one female pup per litter, the brain, spleen and thymus were weighed.  Homogenisation resistant testicular spermatids were counted for the P and F1 parental males and ovarian follicle evaluation was performed for the F1 generation parental females.

There were no test item-related deaths for either the P or F1 generation.  Clinical signs associated with the substance were limited to excessive salivation at the time of dosing for males and females given 50 mg/kg/day for both generations.

For the P generation, the males given 50 mg/kg/day gained slightly less weight than Controls from Week 4 of the study onwards.  The offspring of the P generation weighed slightly less than Controls after birth; the pups gained weight thereafter but at 50 mg/kg/day the animals remained slightly lighter than the Controls.  Consequently, animals in this group were slightly lighter than the Controls at the start of the F1 generation; however, these animals gained weight so that by Week 2 of the F1 generation, body weights were generally comparable across the groups.  There was no adverse effect of the substance on food intake for either generation.  

There was no effect of the substance on oestrous cycling, fertility and mating performance or gestation length for either generation.  Pregnant females gave birth to live pups and there was no effect on the sex ratio.  

The postnatal survival of the F1a animals was reduced at 50 mg/kg/day, with an increased incidence of pup deaths from Day 7 of age to weaning; reduced pup survival was most notable for a small proportion of the litters, with total litter loss for 1 female only.  At necropsy, the absence of milk in the stomach was noted for most of the early decedent pups.  However, this was not repeated in the subsequent generation, and there was no effect on pup survival for the F2a animals.  

At 50 mg/kg/day, sexual maturation of the F1 generation males was approximately 2 days later than Controls, however, individual values were generally comparable with the Controls and the historical control range.  Delays in sexual maturation were not seen for the F1 generation females and there was no effect on anogenital distance for the F2a animals.  

There was no effect of the substance on sperm parameters for males from either generation or on the quantification of the F1 generation ovarian follicles.  

High liver weights were noted in both P and F1 generations given 50 mg/kg/day, which typically correlated with microscopic findings of centrilobular hepatocyte hypertrophy and/or centrilobular hepatocyte vacuolation.  Higher kidney weights were also noted for the P generation males given 50 mg/kg/day and F1 generation males given 10 or 50 mg/kg/day.  The kidneys for the F1 generation males were microscopically examined following macroscopic abnormalities at necropsy, identifying an increased incidence of minimal basophilia in the cortical tubules at 50 mg/kg/day.   Since the finding was minimal and is a relatively common finding, the observation was considered not to be clear evidence of toxicity.  Other organ weight changes for the P and F1 generations were considered to be minimal and not adverse.  For the F1a pups, spleen and thymus weights in the 50 mg/kg/day group were slightly lower than the Controls.  There was no effect on organ weights for the F2a pups.

In conclusion, Administration of the substance, once daily, by oral gavage at 2.5, 10 or 50 mg/kg/day to male and female Crl:WI(Han) rats for two successive generations was generally well tolerated, with no effect on reproductive performance, mating behaviour or conception.  Reduced pup

survival was noted from Day 7 of age to weaning for the offspring of the P generation given 50 mg/kg/day, resulting in a slight reduction in survival for the overall lactation period (lactation index), although this did not achieve statistical significance.  The pups from this dose level were also lighter than Controls after birth and there was a slight increase in the time taken by the males to reach sexual maturation.  There were no similar effects in the offspring of the F1 generation.  Based on these findings, the No-Observed-Adverse-EffectLevel (NOAEL) for effects on reproduction was considered to be 50 mg/kg/day.  

In terms of systemic toxicity, effects were primarily limited to animals given 50 mg/kg/day.  At this dose, there was a slight reduction in body weight gain for the Parental generation males, with higher liver weights than Controls in both generations; this was typically associated with centrilobular hepatocyte hypertrophy and centrilobular vacuolation.  Although increases in liver weight were not observed at 2.5 or 10 mg/kg/day, centrilobular hepatocyte hypertrophy was observed in males from both groups and for females given 10 mg/kg/day in the Parental generation and for males given 10 mg/kg/day in the F1 generation, which was considered to represent an adaptive response.  Higher kidney weights were also noted for the F1 generation males given 50 mg/kg/day with increased incidence of minimal basophilia in the cortical tubules.  This latter finding was minimal and it is relatively common in rats and therefore, was considered not to be clear evidence of toxicity.  On the basis of these findings, the No Observed-Adverse-Effect-Level (NOAEL) for systemic toxicity was considered to be 10 mg/kg/day.