5-bromo-1,3-dichloro-2-fluorobenzene

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

NOAEL for toxicity on sexual function and fertility: 50 mg/kg bw/day (rat, GLP, OECD TG 416, subchronic exposure)

NOAEL for reproductive toxicity: could not be established

LOAEL for reproductive toxicity: 5 mg/kg bw/day (rat, GLP, OECD TG 416, subchronic exposure), based on increased incidence of pup mortality

 

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Remarks:

This study was conducted to meet the data requirements for registration in a non-EEA country requesting such data.

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start: 13 August 2020 (animal arrival), 25 August 2020 (start of dosing)
Experimental end: 30 April 2021 (in-life end), 04 October (signature of pathology report)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Supplier: Charles River (UK) Limited, Margate, Kent, CT9 4LT, United Kingdom
Age: five to six weeks (males) or four to five weeks (females) on arrival
Acclimatisation: 12 days
Weighed: 192 to 264 g (males) and 117 to 175 g (females) on first day of dosing

Housing: in groups, by sex, until pairing and for males post-pairing in solid-floor caging (Techniplast 1500U) with bedding; for pairing one male and one female from the same group were housed together in grid-floor cages suspended over paper-lined trays; mated females housed individually and subsequently with their litter in solid floor cages (NKP RB3) with bedding material
Environment: illumination to give a cycle of 12 hours light and 12 hours darkness, temperature between 20 and 24 °C, humidity between 40 and 70%
Diet: pelleted rodent diet VRF1 (SDS) supplied by Charles River (UK) Limited, Margate at libitum
Water: main tap water in bottles ad libitum

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

The test substance was formulated in a fume hood within the stability period, for each group separately, as a suspension in corn oil. The test substance was inspected for any signs of crystallisation and, if apparent, was warmed to about 30 °C in a water bath for up to 182 minutes until liquid and mixed by swirling. The substance was weighed into a container and the required amount of vehicle was added to make up to final weight. The mixture was stirred until the test item was suitably suspended.
The final suspensions were divided into daily aliquots and stored at room temperature in amber glass dose bottles. They were stirred from at least 15 minutes before the start of dosing until the completion of their use for dosing, to ensure thorough re-suspension and homogeneity, which was checked visually.

Details on mating procedure:

After the pre-pairing dosing period (approximately 10 weeks), each female was paired with a male from the same dose level for up to 14 days. On confirmation of mating, the males were returned to the group cages and the females were housed individually.
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.
During the pairing period, vaginal smears were also taken daily, by lavage, until mating was confirmed by sperm being found in the smear. The number of ejected copulation plugs was recorded to give an assessment of the mating activity of the animals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were taken from each formulation. Sets of samples taken from each formulation prepared for the first day of dosing were analysed to confirm the concentration of the test substance and homogeneity. Thereafter, sets of samples taken from all test item formulations prepared at two monthly intervals were analysed to confirm satisfactory concentration. For Controls, sets of samples (for analysis or for contingency) taken from the vehicle used on the first day of dosing and at two monthly intervals thereafter were analysed to confirm absence of test item.
All tested formulations were considered to have been accurately prepared and homogeneous. No test substance was detected in the vehicle corn oil used to dose control animals on any occasion.

Duration of treatment / exposure:

P and F1 generations: about 18 weeks of exposure; females were exposed 10 weeks prior to mating, 2 weeks during pairing, 3 weeks during gestation, 3 weeks during lactation; males were exposed 10 weeks prior to mating, 2 weeks during pairing, 6 weeks after pairing

Frequency of treatment:

Daily administration

Details on study schedule:

Animals were administered the test item by oral gavage for at least 10 weeks before pairing for mating, and then during and after pairing until the day before necropsy for males and during pairing, gestation and until Day 20 of lactation for females. Animals that were mid parturition at the time of dosing were not dosed on that day. The selected F1 generation were dosed from Day 21 of age. Individual dose volumes were based on the most recently recorded body weight using a dose volume of 4 mL/kg body weight.
For the F1 generation, a high incidence of pup mortality was observed in the test item groups (mainly at 15 and 50 mg/kg/day). Due to the extent of this mortality, all surviving F1 females and F2a pups from the 50 mg/kg/day group (with and without total litter loss) and the surviving F1 generation females with total litter loss from the 5 and 15 mg/kg/day groups were killed prematurely (on Days 3 to 7 of lactation). The F1 generation females that failed to litter were also euthanised in conjunction with the females with total litter loss. Due to the timescales involved in implementing this approach, some of the animals were also dosed on the day of necropsy; this was considered not to have impacted on the pathological assessments.
The F1 generation females with surviving litters from the 5 and 15 mg/kg/day groups, all Control females and males from all groups continued on study, as scheduled and detailed above.

No. of animals per sex per dose:

24 animals per control and dose group

Control animals:

yes, concurrent no treatment

Details on study design:

Dose levels were selected in consultation with the Sponsor based on results from a 14 day non-pregnant dose range-finding study, a developmental toxicity screening study (OECD 422), and the preliminary results of a 90 day study performed at Sequani. In the 14 day dose range-finding study, piloerection was present at 100 and 300 mg/kg/day, as well as a minimal effect on food intake. In the OECD 422 study, females in the
300 mg/kg/day dose group were euthanised by Day 9 due to general physical condition and body weight loss. Females given 150 mg/kg/day also showed significantly lower body weight gain in the first two weeks of the treatment period. In the 90 day study, no test item-related clinical signs or effects on the body weight parameters were observed at any dose level up to 50 mg/kg/day. Based on the above findings, 50 mg/kg/day was selected as the high dose level for this study. The mid and low doses of 15 mg/kg/day and 5 mg/kg/day were calculated by a factor of approximately three.

Positive control:

Not applicable

Parental animals: Observations and examinations:

Mortality, morbidity: Twice daily, individual record
Clinical signs: Daily, individual record
Body weight: Weekly using an electronic balance
Body weight (before mating): Weekly using an electronic balance
Body weight (gestation): Days 0, 7, 14 and 20 of gestation using an electronic balance
Body weight (lactation): Days 0, 1, 4, 7, 14 and 21 of lactation and on the day of necropsy using an electronic balance
Food consumption: Weekly (except during cohabitation for mating) based on the food weighed in and the food weighed out; an electronic balance was used.
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.

All F1 generation females were examined daily from Day 25 of age for vaginal opening. All F1 generation males were examined daily from Day 35 of age for balano-preputial separation. (The balano-preputial separation data identified animals from Control and test item groups, where the day of attainment was later than typically observed in this strain of rat (Day 50 of age or above). A definitive cause for the variance in this subjective end-point could not be confirmed, and therefore, conclusions on any effect of the test item on this parameter could not be drawn. As a result, the group mean summary data were not presented in the final study report).
Body weights were recorded on the day that these indicators of sexual maturation were observed.

Oestrous cyclicity (parental animals):

Daily for 3 weeks premating and throughout cohabitation until evidence of positive mating and on the necropsy day.

Sperm parameters (parental animals):

Sperm motility and concentration were assessed for all P and F1 generation males killed at scheduled necropsy using the Hamilton Thorne 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 10% 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. In the absence of a test item-related effect at the high dose level, sperm morphology was not assessed for the low and intermediate dose groups.

Litter observations:

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, four males and four females. Litters of fewer than eight 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.
Mortality and morbidity: Twice per day
Clinical observations and malformations: Daily
Body weights: Pups were weighed individually on Days 1, 4, 7, 14 and 21 of age
Anogenital distance: The anogenital distance of each surviving F1a and F2a pup was measured on Day 1 of age. Body weights were recorded for the pups at the same time as anogenital distance was measured, to allow calculation of the anogenital distance index
Number of nipples/areolae: For each F1a and F2a male pup, the number of nipples/areolae were counted on Day 12 of age.

Postmortem examinations (parental animals):

Necropsy: For the P generation, females with litters were killed on Day 21 of lactation at weaning of their litters. The remaining females, including those that were apparently not pregnant, those that failed to mate and those with total litter loss were euthanised after confirmation that a second mating was not required.
For the F1 generation, the females with surviving litters at 50 mg/kg/day and all females with total litter loss (from all test item groups) were euthanised prematurely, due to a high incidence of pup mortality; the day of euthanasia equated to Day 3 to 7 of lactation. The non-littering females from all groups were also euthanised in conjunction with these animals, as a second mating was not required. Females with surviving litters at 5 and 15 mg/kg/day, and all Control females, were euthanised on Day 21 of lactation.
The males from both generations were killed approximately six weeks after completion of the mating phase (after successful littering).
A dead body weight was recorded and the cranial, thoracic and abdominal cavities were opened 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.
Organ weights: The following organs were weighed after trimming of fat and other contiguous tissue (contralateral organs were weighed together): adrenals, brain (including olfactory bulbs, epididymides, epididymis cauda, kidneys, liver, ovaries, pituitary, prostate, seminal vesicles (including coagulating gland), spleen, testes, thyroids (including parathyroids), uterus (including uterine cervix and oviducts).
Macroscopic and microscopic pathology: For all animals, with the exception of the brain and testes, either whole organs or samples of the tissues listed below, were preserved in 10 % buffered formalin. The brain and testes were fixed in Modified Davidson’s for 24 to 72 hours before being transferred into 10 % buffered formalin.
Fixated organs: adrenal glands, brain, pituitary, spleen. Fixated and processed organs: epididymides, kidneys, liver, mammary gland, ovaries, prostate, seminal vesicles (including coagulating gland), testes, thyroids (including parathyroids), uterus (including uterine cervix and oviducts), vagina, all gross lesions.
P generation: For all animals, the tissues specified above 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 and any premature decedents initially, the tissues specified in the tissue list were examined microscopically. Additionally, reproductive organs of the low and intermediate dose groups 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. Following the initial pathology review, the kidneys, liver and thyroids for all males were processed to slide and examined microscopically. The vagina and mammary tissue for the low and intermediate group females were also examined microscopically. An external peer review was performed by the Sponsor’s pathologist.
F1 generation: For all animals, the tissues specified above 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 and any premature decedents, the tissues specified in the tissue list were examined microscopically. Additionally, reproductive organs of the low and intermediate dose groups suspected of reduced fertility (i.e. 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. Following the initial pathology review, the kidneys and liver for all low and intermediate group males, and the liver for all females, were processed to slide as detailed above and microscopically examined. In addition, the reproductive organs for the low and intermediate dose group females (uterus, cervix, vagina, ovaries and oviducts; and the mammary tissue were also examined microscopically to aid further assessment of the study results. An external peer review was performed by the Sponsor’s pathologist.

Postmortem examinations (offspring):

Necropsy: 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 surviving pups on Day 21 of age; this included a necropsy for all high dose group F2a pups following the premature euthanasia of the F1 generation females and F2a pups from this group. 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). A dead body weight was recorded for pups where organ weights were to be determined on Day 21 of age. The cranial (for pups on Day 21 of age only), thoracic and abdominal cavities were opened 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.
Organ weights: For one male and one female pup from each litter euthanised on Day 21 of age, the following organs were weighed after trimming of fat and other contiguous tissue: brain (including olfactory bulbs) spleen, thymus.
Macroscopic pathology: For one male and one female pup per litter euthanised on Day 21 of age (the same pups assigned to organ weight measurement), the tissues listed below were retained. All other pups had a gross macroscopic examination only and gross abnormalities were retained. With the exception of the brain, either whole organs or samples of the tissues listed below were preserved in 10% buffered formalin. The brain was fixed in Modified Davidson’s solution before being transferred into 10% buffered formalin: 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 10% buffered formalin.

Statistics:

See below "Any other information on materials and methods"

Reproductive indices:

Female copulation index (%) = # females mated/# females paired x 100
Male copulation index (%) = # males mated/# males paired x 100
Female fertility index (%) = # pregnant females/# mated females x 100
Male fertility index (%) = # males siring a pregnancy/# males paired x 100
Gestation index (%) = # pregnant females with live pups born/# pregnant females x 100
Post-implantation loss (%) = (# implantation scars - # pups born)/# implantation scars x 100
Anogenital distance index ADGI (%) = anogenital distance/cubed root of individual pup body weight x 100

Offspring viability indices:

Live birth index (%) = # pups born alive/# pups born x 100
Viability index 1 (%) = # pups alive Day 4 before culling/# pups born alive x 100
Viability index 2 (%) = # pups alive Day 7/# pups alive Day 4 after culling x 100
Viability index 3 (%) = # pups alive Day 14/# pups alive Day 7 x 100
Viability index 4 (%) = # pups alive Day 21/# pups alive Day 14 x 100
Lactation index (%) = # pups alive Day 21/# pups alive Day 4 after culling x 100
Cumulative survival index (%) = (# pups Day 21/# pups Day 4 after culling) x (# pups Day 4 before culling/# pups born) x 100

Clinical signs:

no effects observed

Description (incidence and severity):

At 50 mg/kg/day, total litter loss was seen in two females (Females 179 and 186); there were no signs of toxicity in these dams and so they were retained until the end of the lactation phase.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males treated with 15 or 50 mg/kg bw/day had slightly lower weight gains than the controls throughout the dosing period. The reduction was minimal, not statistically significant and the absolute group mean body weights for the males were only 2% and 4% lower at the end of the dosing period. The finding was considered to be non-adverse.
Body weights and body weight gains were unaffected by exposure to the substance during the pre-paring period. During gestation, a slight dose-related reduction in group mean weight gain was seen in all groups dosed with the substance for the overall period when comparing with controls. This reduction was statistically significant at 50 mg/kg bw/day. It was primarily due to reduced weight gain from Day 14 of gestation, with the intergroup differences exacerbated by high individual variation in the controls. The body weight gains during lactation were comparable with or higher than the controls. The slight and transient reduction in maternal body weight during late gestation was therefore considered to be non-adverse.
At 15 or 50 mg/kg/day, the P1a pups were slightly, but statistically significantly, lighter than the Controls after birth (Day 1 of age: up to 11% lower for male and female pups combined; p≤0.01). There was no difference between the dose groups and relatively low pup weights seen in both litters with and without reduced pup survival. Although the surviving pups gained weight thereafter, the overall weight gain for the lactation period in these groups remained slightly lower than the Controls (5% or 7%, respectively), so that the mean weights were up to 7 % lower at weaning (Day 21 of lactation: p≤0.05 at 50 mg/kg/day).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In males, a marginal, non-statistically significant reduction in food intake was seen over the first week of dosing at 50 mg/kg/day compared with Controls (7% lower). Since values were comparable with the Controls thereafter, the initial reduction was considered to be non-adverse.
In females, food intake was unaffected by exposure to the substance throughout the pre-pairing and gestation periods. During the first week of lactation only, group mean food intake for females given 50 mg/kg/day was slightly lower than the Controls, particularly over Days 4 to 7 of lactation (13% lower; p≤0.05). There was a large degree of individual variation within this group, with the lowest food intake generally coinciding with the females with small litters (such as Female 174) and those with total litter loss (Female 186; food intake measurements were excluded from group mean values after the day of total litter loss). This initial reduction at 50 mg/kg/day was considered non-adverse as food intake was comparable across the groups from Day 7 of lactation.

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

no effects observed

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):

In the males, findings considered to be related to the substance were seen at 15 or 50 mg/kg/day in the kidneys (increased hyaline droplets) and thyroid (hypertrophy of follicular epithelium and increased basophilia of the colloid). In addition at 50 mg/kg/day only, basophilia of the cortical tubules and epithelial necrosis/distension of cortico-medullary tubules was seen in the kidney and there was centrilobular hypertrophy in the liver. The findings in the thyroid correlated with corresponding organ weight increases. These changes were all considered to be either rat specific and/or adaptive and therefore non-adverse.
In the females, findings considered to be related to treatment were seen in the mammary gland (reduced lactational activity) and in the female reproductive system, as evidenced by higher numbers of animals in lactational dioestrus (50 mg/kg/day) and a higher number of mature corpora lutea in the ovaries, particularly at 50 mg/kg/day. Reduced lactational activity was expected for the two females with total litter loss (Females 179 and 186, severe and markedly reduced, respectively) as the pups died before the parental females were euthanised; however, reduced lactational activity was also noted in two females that maintained pup survival until weaning (Females 174 and 192). It should be noted that the majority of animals had returned to oestrous cycling by the time of termination, albeit at slightly differing stages.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

There were no microscopic findings to indicate a cause for the apparent infertility in the animals that failed to mate or those that mated, but mating did not result in pregnancy. Papillomatous hyperplasia in the uterus and muco-fibrinous contents in the vagina was noted for one of the 50 mg/kg/day females which failed to mate (Female 190), however, this isolated observation was considered not to be test item-related.

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no differences in sperm motility, velocity or concentration for the P generation males exposure to any dose level of the substance when compared with controls. At 50 mg/kg bw/day, sperm morphology was unaffected by treatment with the substance and homogenisation resistant testicular spermatid counts were similar to controls.
Anogenital distance and nipple counts in pups: For all dosed groups, there was a dose-related increase in the mean anogenital distance index for male and female pups compared with Controls (AGDI: anogenital distance adjusted to the cubed pup body weight); the difference was statistically significant in males at 15 or 50 mg/kg/day (16% higher; p≤0.01) and in females at 50 mg/kg/day only (25% higher; p≤0.05). This was also reflected in a slight increase in absolute anogenital distance in the males and females, despite the comparatively low pup body weights seen in these groups. The AGDI was higher than the historical Control range for males at 15 or 50 mg/kg/day only. This was not replicated in the surviving pups from the next generation (F1a pups) and therefore, the toxicological significance of this finding is unclear. There were no retained nipples or areolae on Day 12 of age in the males from any group treated with the substance.

Reproductive performance:

no effects observed

Description (incidence and severity):

At 15 and 50 mg/kg/day, the male and female copulation indices were slightly lower than the Controls, due to one female (Female 157) and two females (Females 176 and 190) in each of these respective groups failing to mate with their allocated male (Males 61, 80 and 94, respectively). This finding was considered to reflect normal biological variation and not to be associated with exposure to the substance, as the indices were either within (15 mg/kg/day) or only marginally below (50 mg/kg/day) the historical Control range.
Normal oestrous cyclicity was seen in all three of these females before pairing, however, persistent dioestrus was observed during the pairing period which may have inhibited copulation. Aside from the papillomatous hyperplasia in the uterus and muco-fibrinous contents in the vagina for Female 190, which were considered not to be test item-related, there were also no pathology findings in either the males or Females 157 or 176 that would have inhibited mating. The pre-coital interval for the mated animals was comparable across the groups, with most animals mating within four days.
There were 20, 23, 21 and 22 pregnant females in the Controls and groups exposed to 5, 15 or 50 mg/kg/day, respectively. Although the pregnancy rate for the Controls was lower than that typically seen in this type of study, most of the mated females given the test substance were pregnant, thereby confirming the absence of an effect on fertility.
Gestation and parturition: There was no effect of the substance on the mean duration of gestation and all pregnant females gave birth to live litters.
Pregnancy and litter data: There were 20, 23, 21 and 22 females with litters in the Control group and groups given 5, 15 or 50 mg/kg/day, respectively. There were no effects on the implantation data that were attributed to the substance. At 50 mg/kg/day, the mean number of pups born per litter was slightly lower than the Controls. This was, however, attributed to normal biological individual variation, as a low number of uterine implantations was only seen in one female (Female 179 with three implants) and marginal increases in post-implantation above the variation seen in the Controls were limited to only two females (Female 171 and 183).
At 50 mg/kg/day, there were fewer pups born alive and postnatal survival was lower than the Controls in the initial days after parturition, particularly over Days 0 to 1 of lactation; this was reflected in a reduced live birth index (p≤0.05) and viability index 1 (not statistically significant). Although pup survival was generally comparable with the Controls from Day 4 of age onwards, the overall cumulative survival index (birth to weaning) remained lower than the Controls (23% lower; p≤0.01). This result was attributed to increased pup mortality in a relatively small number of litters, rather than a systemic effect across the group; there were only five litters with a live birth index less than 70% (Females 177, 179, 182, 183, 192) and four litters with viability index 1 (birth to Day 4 of age) of less than 70% (Females 174, 175, 179, 186). Whilst total litter loss was seen for two of these females by Day 4 of age (Females 179 and 186), the remaining females successfully reared their surviving pups to weaning. The cause for the reduced survival was not clear, however, the clinical condition of the pups during the initial days of lactation may have been a contributing factor.
There was no effect on the mean number of pups born alive or on the postnatal survival at 5 or 15 mg/kg/day, and no effect on the pup sex ratio at any dose level.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

female reproductive system

Organ:

mammary gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

presumably yes

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no abnormal clinical signs that were attributed to the substance throughout the P1 generation.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Females with total litter loss were euthanised prematurely. This was most pronounced at 50 mg/kg/day, where the death of all pups in the litters of 15 of the 21 pregnant females in the days after parturition (typically Days 0 to 2 of lactation) resulted in the euthanasia of all surviving females and litters in this group between Days 3 to 6 of lactation. The incidence of total litter loss was less pronounced at 5 or 15 mg/kg/day, affecting one or nine females in each group, respectively; as a result, the remaining females in these groups were allowed to rear their offspring to Day 21 of lactation as planned.
Reduced maternal weight gain in the latter stages of gestation was seen, however, there were no other signs of toxicity for most females to clearly indicate a cause for the pup mortality; the exception to this was for one female at 50 mg/kg/day (Female 388), which was euthanised the day after total litter loss (Day 1 of lactation) due to a decline in its clinical condition (signs of piloerection and loose faeces). There were no test item-related macroscopic findings in these decedent females, however, reduced lactational activity in the mammary gland was seen microscopically in most of the females with total litter loss and in some individuals with surviving litters from the 50 mg/kg/day group; atypical lactational dioestrus was also observed at 15 and 50 mg/kg/day (including females with and without surviving litters at 50 mg/kg/day). Since most females were euthanised several days after the pups had died, the relevance of these findings is unclear.
One female at 50 mg/kg/day (Female 392), was euthanised on Day 23 of gestation following signs of dystocia, which was confirmed at necropsy by the presence of pup in utero. This isolated occurrence was not attributed to treatment with the substance, as difficulties in parturition were not seen in any other female.
At 15 mg/kg/day, one male (Male 250) was euthanised on Day 89 of the P1 generation (after pairing) due to a 16% body weight loss within five days during Week 12 (value not reported). At macroscopic necropsy, the liver was abnormally pale, red areas were noted on the deep axillary lymph nodes and the thymus and pancreas were abnormally dark. Also at 15 mg/kg/day, one female (Female 345) was euthanised on Day 14 of gestation due to a hunched posture and piloerection on Days 12 to 14 of gestation and a 13% body weight loss over Days 0 to 14 of gestation. The ovaries were large at macroscopic necropsy and the thymus was small. There were no microscopic changes in either animal to indicate a cause for their decline. In the absence of similar findings in the remaining animals, these were considered to be isolated instances that were unrelated to treatment with the substance.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 15 or 50 mg/kg/day, the males and females were slightly lighter than the Controls at the start of the P1 generation.
In males thereafter, body weight gains were generally comparable across the groups, so that the mean weights were broadly similar at the end of the study.
In females, body weight gains over the pre-pairing period in the dosed groups were comparable with, or slightly higher than Controls; the increase in weight gain at 50 mg/kg/day was statistically significant (Weeks 0 to 10: 11% higher; p≤0.01).
During gestation, however, group mean body weight gains for females in all treated groups were lower than the Controls from Days 14 to 20 of gestation, resulting in statistically significantly lower body weight gains for the overall period (Days 0 to 20 of gestation: 19%, 24%, 24% at 5, 15 or 50 mg/kg/day, respectively; p≤0.01). Particularly low weight gains were often observed in the females with subsequent total litter loss, however, this was not a consistent trend.
In contrast, compensatory increases in body weight gain were seen at the start of the lactation phase for all groups and continued until at least Day 14 of lactation for the surviving females, so that body weights were comparable with Controls at 5 or 15 mg/kg/day by Day 21 of lactation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food intake in the males throughout the study, and in the females before pairing, was unaffected by treatment with the substance, with any slight, but statistically significant intergroup differences considered to reflect normal biological variation.
During gestation, food intake was comparable across the groups until Day 14 of gestation. Thereafter, however, mean food intake for females in all treated groups was statistically significantly lower than the Controls (p≤0.01); from Days 14 to 17 of gestation, the degree of the reduction was comparable across the dosed groups (up to 15% relative to Controls), but between Days 17 and 20 of gestation, the decrease was dose-related (22%, 26%, 32% lower than Controls, respectively).
At the start of lactation, group mean food intake was lower than the Controls for the females at 15 mg/kg/day (17% lower; p≤0.05) or 50 mg/kg/day (22% lower); limited lactational food intake was available at 50 mg/kg/day due to the euthanasia of all females from the group. Whilst increases were seen at 15 mg/kg/day thereafter, values remained lower than Controls, so that the overall mean intake was 14% lower from Day 1 to 21 of lactation (p≤0.05). At 5 mg/kg/day, food intake was generally comparable with the Controls throughout lactation.

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

Endocrine findings:

no effects observed

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):

In males given 15 or 50 mg/kg/day, there was a dose-related increase in mean kidney weights compared with Controls (adjusted values: 8% or 21%, respectively; p≤0.01). This correlated with the increase in hyaline droplets seen microscopically at these dose levels.
In males, group mean liver weights were higher than the Controls at all dose levels of the substance. The highest weights were seen at 50 mg/kg/day (approximately 30% higher: p≤0.01), where the body weight-related values for most males exceeded the upper limit of the normal historical Control range. There was no clear difference between 5 or 15 mg/kg/day. This finding correlated with centrilobular hypertrophy in the liver at 15 mg/kg/day (minimal) and 50 mg/kg/day (slight).
There were no other organ weight changes that were attributed to exposure to the substance. This included the apparent slightly lower mean absolute brain weight and increase in thyroid weight for males at 50 mg/kg/day (p≤0.01 or p≤0.05, respectively) and the slight increases in prostate weight and seminal vesicle weights in all treated groups (prostate: p≤0.05 at 15 mg/kg/day, seminal vesicles: p≤0.05 at 50 mg/kg/day). These apparent intergroup differences were instead considered to reflect normal biological variation, as the weights for most individuals remained within the variation seen in the concurrent Controls.
There were no substance-related organ weight changes in the females on Day 21 of lactation. The organ weights for the females at 50 mg/kg/day and the weights for the females with total litter loss could not be directly compared with the remaining females due to the differences in the day of euthanasia, however, there were no clear organ weight changes in these females to indicate an effect of the substance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no macroscopic findings that indicated a clear effect of the substance. Although microscopic changes were subsequently observed in the kidneys and liver for the males at 15 or 50 mg/kg/day and the liver for females at 50 mg/kg/day, these organs were macroscopically normal for most animals.
A variety of spontaneous changes was noted in Control and treated animals with no indication of an effect of treatment. The spectrum of these findings was generally consistent with changes encountered in rats of this age kept under laboratory conditions.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the males, findings considered to be related to treatment were seen in the kidneys at 15 or 50 mg/kg/day (basophilia cortical tubules, increased hyaline droplets and epithelial necrosis/distension of cortico-medullary tubules) and liver (centrilobular hypertrophy). The liver changes reflected the higher organ weights recorded; there was no clear microscopic correlate for the increase in kidney weights.
The female livers from all dose groups were examined as a consequence of the high dose gross findings (“pale” which corresponded with hepatocyte vacuolation), with no further evidence of hepatocyte vacuolation beyond the two animals examined initially.
In the females, findings considered to be related to treatment were seen principally at 15 or 50 mg/kg/day in the mammary gland (reduced lactational activity) and in the female reproductive system, as evidenced by the higher number of animals in lactational dioestrus in the mid and high dose groups (15 and 50 mg/kg/day); this also included the presence of atypical lactation dioestrus in both of these doses. This atypical lactational dioestrus was typified by an epithelial layer of mucified cells in the vagina interspersed with vacuoles, containing what appeared to be cellular debris.
By the end of this period of the study the remaining animals had returned to oestrous cycling, albeit at slightly differing stages.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

There were no microscopic findings to indicate a cause for the apparent infertility in the animals that mated, but mating did not result in pregnancy.
There were higher numbers of mature corpora lutea in the ovaries, which are considered to be corpora lutea of pregnancy, consistent with the early sacrificed females (those with total litter loss and those euthanised following premature termination of the 50 mg/kg/day females). Similarly, the increase in mature corpora lutea (typified by the presence of plump, highly luteinised luteal cells) may be indicative of a rat post parturition process not normally examined in a regulatory study.
The presence of uterine arteritis is considered to represent early regression of the implantation sites in animals with shortened lactation periods (early sacrifice).

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There was no effect of exposure to the substance on either the mean number of oestrous cycles or the mean oestrous cycle length during the pre-pairing period.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no differences in sperm motility or concentration for the F1 generation males that were considered to be related to treatment with the substance at any dose level. At 15 mg/kg/day, the slight increase in mean sperm concentration and velocities compared with Controls (p≤0.01), which were attributed to normal biological variation and not to be associated with exposure to the substance, in the absence of a similar effect at 50 mg/kg/day.
Sperm morphology was unaffected by treatment with the substance and homogenisation resistant testicular spermatid counts for males given 50 mg/kg/day of the test substance were similar to Controls.

Reproductive performance:

no effects observed

Description (incidence and severity):

There was no effect of exposure to the substance on copulation or fertility at any dose level.
All animals exposed to the substance mated, with most mating within the first four day of pairing.
The fertility indices for the groups given the substance were slightly lower than the Controls, as three, two and two females in the groups given 5, 15 or 50 mg/kg/day, respectively, were not pregnant (Females 324, 325 and 333 at 5 mg/kg/day, Females 345 and 353 at 15 mg/kg/day and Females 377 and 380 at 50 mg/kg/day). There were no microscopic changes in these females or the pairing males (Males 240, 241, 225, 261, 269, 293, 296) to indicate a cause for the apparent infertility. Since at least 21 of the 24 females in each group were pregnant, the pregnancy rates were comparable with the Controls from the parental generation of this study and the fertility indices at 15 or 50 mg/kg/day were within the historical Control range, the apparent slight reduction in fertility was considered not to be associated with the substance.
Gestation and parturition: The mean gestation length was comparable across the groups. At 50 mg/kg/day, only 17 of the 22 pregnant females gave birth to live pups, resulting in a statistically significant decrease in the gestation index compared with the Controls (23% lower; p≤0.01). This was due to three females giving birth to dead pups (Females 372, 389 and 391), one female with no pups remaining after parturition was complete (Female 388 and one female showed signs of dystocia (Female 392). The time to complete parturition was considered not to be a contributing factor, with no signs of elongated parturition. There was no effect on parturition at 5 or 15 mg/kg/day, with all pregnant females giving birth to live pups.
Pregnancy and litter data: There were 24, 21, 22 and 21 females with litters after parturition in the Control group and
groups given 5, 15 or 50 mg/kg/day, respectively. In addition, one female at 50 mg/kg/day (Female 388) was noted to have completed parturition, but there were no pups remaining within the cage.
There was no effect of the substance on the mean number of implantations. The mean incidence of post-implantation loss was marginally higher than the Controls for all treated groups, due to slight increases in implantation loss for some individuals.
At 50 mg/kg/day, there a significant decrease in the number of live pups on Day 1 of age relative to the number of pups born compared with the Controls (live birth index: 28% lower; p≤0.01). Further pup deaths were seen on subsequent days, to the extent that total litter loss was seen in 15 of the 21 littering females by Day 2 of lactation. Although six remaining females had surviving pups at the time all females and surviving litters were euthanised (Days 3 to 6 of lactation), pup survival to Day 4 of lactation was substantially lower than the Controls (viability index 1: 79% lower; p≤0.01).
Lower postnatal survival was also seen in the pups at 5 or 15 mg/kg/day up to Day 4 of lactation (viability index 1: 9% and 53% lower than Controls, respectively; p≤0.01 at 15 mg/kg/day). Although less prominent than the high dose, the reduced survival resulted in total litter loss for one female at 5 mg/kg/day and nine females at 15 mg/kg/day. From Day 7 of age onwards, there was no further pup mortality in the 5 or 15 mg/kg/day groups, however, the initial pup mortality resulted in lower survival indices for the overall lactation period (cumulative survival index: 7% and 54% lower than Controls, respectively; p≤0.01 at 15 mg/kg/day).
There were, on average, fewer male pups per litter at 15 or 50 mg/kg/day compared with the Controls (% males: p≤0.01 at 50 mg/kg/day). This was considered not to indicate a clear effect of the substance, as many of the pups from these groups had died before sexing on Day 1 of age, and therefore, a full assessment on the mean sex ratio could not be made.

Sexual development observations: Sexual development was considered to be unaffected by exposure to the substance at all dose levels.
An unusual degree of ambiguity in the data for male sexual development assessments was recorded, with some individual animals from all groups attaining this milestone at a later time-point than expected. The degree of ambiguity in all groups was such that it was not possible to interpret the male sexual development in this study.
A slight delay (1.3 days) in the mean day of vaginal opening was apparent at 50 mg/kg/day compared with the Controls (p≤0.05); there was no significant difference in the mean body weight on the day of attainment. This was, however, also considered to reflect normal biological variation as the group mean remained within the historical Control range and the day of attainment for only three females exceeded the concurrent Control range (>33 days).
Ovarian follicle quantification: There were no changes in the ovarian follicles that were attributed to exposure to the substance. At 50 mg/kg/day, the mean number of primordial and primary follicles and thus total number of small follicles was slightly lower than the Controls; the mean total number of primordial follicles and small follicles was also below the historical Control range. These intergroup differences were, however, considered to be a consequence of normal inter-animal variation

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

female reproductive system

Organ:

mammary gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

presumably yes

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 15 or 50 mg/kg/day, the number of litters with pups showing a flaky surface to the skin was higher than the Controls in the initial days after birth (generally up to Day 2 of age). Pups with red or purple areas, or a red or pink appearance to the entire body, were also noted from different litters, although less frequently. From Day 3 of age onwards, the affected pups had generally returned to a normal appearance and there was no further effect on their clinical condition. There were no clinical signs in the pups following maternal administration at 5 mg/kg/day.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

At 50 mg/kg/day, there was an increased incidence of pup mortality during the initial days of lactation compared with the Controls (primarily over Days 0 to 2 of lactation); this resulted in the death of a total of 55 pups from this group (found dead, euthanised prematurely or presumed to have been cannibalised), compared with 13 pups in the Controls. In most cases, the pup mortality per litter was low, however, the litters for four females were significantly reduced for the remaining lactation period (five or more pup deaths for Females 174, 175, 182 and 192) and total litter loss was seen for two females with relatively small litters after parturition (Females 179 and 186). There was no effect of maternal administration of the substance on the postnatal survival of the F1a pups at 5 or 15 mg/kg/day.

Body weight and weight changes:

effects observed, treatment-related

Anogenital distance (AGD):

effects observed, treatment-related

Description (incidence and severity):

For all dosed groups in the F1a generation, there was a dose-related increase in the mean anogenital distance index for male and female pups compared with Controls (AGDI: anogenital distance adjusted to the cubed pup body weight); the difference was statistically significant in males at 15 or 50 mg/kg/day (16% higher; p≤0.01) and in females at 50 mg/kg/day only (25% higher; p≤0.05). This was also reflected in a slight increase in absolute anogenital distance in the males and females, despite the comparatively low pup body weights seen in these groups. The AGDI was higher than the historical Control range for males at 15 or 50 mg/kg/day only. As this finding was not replicated in the surviving pups from the next generation (F2a pups) and therefore, the toxicological significance of this finding is unclear.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

There were no retained nipples/areolar on Day 12 in the male pups from the surviving litters.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no organ weight changes that were considered to be directly related to maternal administration of the substance. Group mean spleen weights were slightly lower than the Controls in F1a females only at 15 mg/kg/day and in males and females at 50 mg/kg/day; the reduction in adjusted weights for the F1a females at 50 mg/kg/day was statistically significant ( p≤0.01). These apparent differences were, however, attributed to individual variation, since values ≤0.05 g lower than the variation seen in Controls were limited to only one pup/sex in each of the affected groups (female pups from the litters of Females 146 and 169 and a male pup from the litter of Female 174). Since most values were comparable with the concurrent Controls, the slight intergroup differences were considered to reflect normal biological variation and not to be associated with maternal administration of the substance. Group mean brain and thymus weights were comparable with Controls at all dose levels.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

In the affected F1a litters with high mortality, the pups that were born alive had a cold body surface, hunched posture, small amount of milk or no milk in the stomach, and/or a flaky surface to the body; instances of abnormally purple coloured areas to the body were also seen in the litter of Female 186 (limbs and head). Macroscopic necropsy did not reveal a cause for the pup mortality, with findings limited to no milk in the stomach for most animals. There were no macroscopic necropsy findings in the F1a pups at the end of lactation that were attributed to maternal administration of the substance.

Histopathological findings:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1a

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

Critical effects observed:

no

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Where pups showed clinical signs before death, these were characterised by small amounts or no milk in the stomach and/or cold body surface, with a flaky surface to the skin (similar to that observed in the F1a pups) seen in one litter only (Female 385).

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Maternal administration of the substance was associated with pup mortality during the initial days after parturition at all dose levels.
At 50 mg/kg/day, there was significant pup mortality in the initial days after parturition, with pups from most litters either found dead or were euthanised due to poor clinical condition; this included a high number of pups that were dead after parturition was complete (Day 0). The extent of pup mortality over Days 0 to 2 of age resulted in total litter loss for 15 of the 21 littering females in the group and therefore, the remaining surviving F1 females and F2a pups in this group
were euthanised (between Days 3 and 6 of lactation); this included the six dams and litters which had survived up to a maximum of Day 6 of lactation.
At 5 or 15 mg/kg/day, pup mortality was also seen up to Day 4 of age, in a dose-related manner. Although one female at 5 mg/kg/day and nine females at 15 mg/kg/day had total litter loss, and similar clinical signs were seen to the high dose group, the remaining litters did survive to Day 21 of age.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was a dose-related and statistically significant decrease in group mean pup body weight after birth, with pups on average 9%, 20% and 32% lighter than the Controls on Day 1 of age at 5, 15 or 50 mg/kg/day, respectively (male and female pups combined; p≤0.01). Lower pup weights were not limited to those litters with a high incidence of pup mortality, with relatively low weights seen for most litters in the dosed groups on Day 1 of age. Whilst the surviving pups at 5 or 15 mg/kg/day gained weight thereafter, the mean pup body weight on Day 21 of age at 15 mg/kg/day remained 8% lower than the Controls.

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):

no effects observed

Description (incidence and severity):

There was no effect on the mean anogenital distance index for the surviving male and female pups in the F2a generation, with comparable group means between the Controls and dosed groups. For the 50 mg/kg/day group, data were only available for a limited number of litters due to the extent of pup mortality and therefore, a full assessment at this dose level could not be made.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

There were no retained nipples/areolar on Day 12 in the male pups from the surviving litters.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There were no effects on the organ weights for the F2a pups that were considered to be directly related to treatment with the substance. On Day 21 of age, group mean brain weights for the male and female pups at 5 or 15 mg/kg/day were slightly lower than the Controls. The reduction was not dose-related, with the lowest weights seen at 5 mg/kg/day (absolute: 5% or 3% lower ; p≤0.01 or p≤0.05 for males and females, respectively). This difference reflected the lower body weight for the pups in the treated groups from birth and therefore lower brain weight, rather than a direct effect on brain weight, as the body weight related values were comparable with Controls. There was no effect on thymus or spleen weights following maternal administration of the substance.

Description (incidence and severity):

Macroscopic necropsy of dead pups in all dose groups could not confirm the cause for the pup mortality due to the extent of autolysis and cannibalisation, however, there was no milk in the stomach for most of the decedent pups. There were no treatment-related necropsy findings in the F2a generation pups on Day 21 of age.

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

LOAEL

Generation:

F2a

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Critical effects observed:

no

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

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

Relevant for humans:

presumably yes

Table 1: Treatment related findings during microscopic pathology in P-generation

		Males				Females
Group		1	2	3	4	1	2	3	4
Dose level (mg/kg/day)		0	5	15	50	0	5	15	50
# of rats examined		24	24	24	24	24	24	24	24
Kidneys
Basophilia, cortical tubules	Minimal	3	2	0	11	-	-	-	-
	Slight	0	0	0	9	-	-	-	-
	Total	3	2	0	20	-	-	-	-
Increased hyaline droplets	Slight	0	0	4	6	-	-	-	-
	Moderate	0	0	0	14	-	-	-	-
	Marked	0	0	0	4	-	-	-	-
	Total	0	0	4	24	-	-	-	-
Epithelial necrosis & distension of cortico-medullary tubules	Minimal	0	0	0	1	-	-	-	-
	Slight	0	0	0	3	-	-	-	-
	Moderate	0	0	0	11	-	-	-	-
	Marked	0	0	0	4	-	-	-	-
	Total	0	0	0	19	-	-	-	-
Liver
Centrilobular hypertrophy	Minimal	0	0	0	16	-	-	-	-
	Slight	0	0	0	8	-	-	-	-
	Total	0	0	0	24	-	-	-	-
Thyroid gland
Hypertrophy of follicular epithelium	Minimal	0	1	6	6	-	-	-	0 a
	Slight	0	0	0	2	-	-	-	0 a
	Total	0	1	6	8	-	-	-	0 a
Increased basophilia, colloid	Minimal	2	4	10	11	-	-	-	0 a
	Slight	0	0	1	0	-	-	-	0 a
	Total	2	4	11	11	-	-	-	0 a
Mammary gland
Reduced lactational activity	Slight	0	-	-	0	0	0	1	0
	Marked	0	-	-	0	0	0	0	2
	Severe	0	-	-	0	0	0	0	2
	Total	0	-	-	0	0	0	1	4
Vagina (stage of oestrous)
	Proestrus	-	-	-	-	4	1	3	3
	Lactational dioestrus	-	-	-	-	1	2	1	6
	Oestrus	-	-	-	-	2	2	6	7
	Metoestrus	-	-	-	-	1	4	5	2
	Dioestrus	-	-	-	-	14	15	9	5

a only one animal examined

Table 2: Treatment related findings during microscopic pathology in P1-generation

		Males				Females
Group		1	2	3	4	1	2	3	4
Dose Level (mg/kg/day)		0	5	15	50	0	5	15	50
Number of rats examined		24	24	24	24	24	24	24	24
Kidneys
Basophilia, cortical tubules	Minimal	5	4	5	19	0	-	-	0
	Slight	0	0	0	3	0	-	-	0
	Moderate	0	0	0	2	0	-	-	0
	Total	5	4	5	24	0	-	-	0
Increased hyaline droplets	Slight	0	1	8	11	0	-	-	0
	Moderate	0	0	0	13	0	-	-	0
	Total	0	1	8	24	0	-	-	0
Epithelial necrosis/distension of cortico-medullary tubules	Minimal	0	0	0	2	0	-	-	0
	Slight	0	0	2	11	0	-	-	0
	Moderate	0	0	0	4	0	-	-	0
	Marked	0	0	0	2	0	-	-	0
	Total	0	0	2	20	0	-	-	0
Liver
Centrilobular hypertrophy	Minimal	0	0	6	24	0	0	0	21
	Total	0	0	6	24	0	0	0	21
Mammary gland
Reduced lactational activity	Slight	0	-	-	0	0	2	9	8
	Moderate	0	-	-	0	0	0	2	4
	Marked	0	-	-	0	0	0	0	7
	Total	0	-	-	0	0	2	11	19
Vagina (state of oestrous)
	Atypical lactational dioestrus	-	-	-	-	0	0	9	7
	Lactation dioestrus	-	-	-	-	5	4	3	9
	Proestrus	-	-	-	-	2	7	1	0
	Oestrous	-	-	-	-	5	5	4	0
	Metoestrus	-	-	-	-	1	2	3	0
	Dioestrus	-	-	-	-	10	6	4	8

Table 3: Findings in the ovaries and uterus of females in P1 generation

		Females
Group		1	2	3	4
Dose Level (mg/kg/day)		0	5	15	50
Number of rats examined		24	24	24	24
Ovaries
Increase in mature corpora lutea	Minimal	0	1	0	0
	Slight	0	0	7	10
	Moderate	0	0	0	9
	Total	0	1	7	19
Uterus
Arteritis, implantation sites	Minimal	0	1	0	1
	Slight	0	0	1	5
	Moderate	0	0	2	4
	Total	0	0	3	10

 

Conclusions:

The NOAEL for parental systemic toxicity in the two-generation reproductive toxicity study was 5 mg/kg bw/day. Exposure to the substance had no adverse effect on fertility parameters, including oestrus cycle and sperm quality measurements. However, increased pup mortality occurred in the second generation in all treated groups in a dose-dependent manner. Thus, the LOAEL for reproductive effects in this study was 5 mg/kg bw/day.

Executive summary:

A two-generation reproductive toxicity study was conducted under GLP to OECD TG 416 with the substance. Four groups of 24 male and female rats of the Crl:WI(Han) strain were dosed with the test substance by oral gavage at dose levels of 0 (corn oil as vehicle control), 5, 15 or 50 mg/kg bw/day for ten weeks before pairing, during pairing, gestation and laction and until necropsy (parental generation). Four groups of 24 male and female P1 generation animals were selected from the weaned P generation litters. These animals were dosed once daily by oral gavage from Day 21 of age at the same dose levels as P generation, with the aim that all P1 generation animals would be dosed for 10 weeks before pairing, during pairing, gestation and lactation until necropsy. However, the surviving females and litters in the group dosed at 50 mg/kg bw/day were euthanised within the first week of lactation due to impaired pup survival. Pup mortality was also seen at 5 and 15 mg/kg bw/day, although not to the extent that the viability of the group was compromised.
The oral administration of the substance did not cause mortality in the parental animals of P and P1 generations. One female given 50 mg/kg bw/day from the P1 generation was euthanised in late gestation due to signs of dystocia. One male and one female in the P1 generation receiving 15 mg/kg bw/day were euthanised following body weight loss and/or onset of clinical signs (after pairing and on Day 14 of gestation). These findings were considered not to be treatment related.
There were no overall adverse effects on body weight in the adult animals in the P generation, with only a slight reduction in body weight gain seen in the males at 15 and 50 mg/kg bw/day over the dosing period and in the females at all dose levels during late gestation. At the start of the P1 generation, animals dosed at 15 or 50 mg/kg bw/day were lighter than control animals, but the body weight in these groups was back to normal during the pre-pairing period for the females and throughout the study for the males. Reduced body weight gain was also seen in the P1 generation females in late gestation. The effect was mor pronounced than in the P generation animals and was considered to have potentially contributed to the increased pup mortality in the F2a generation. Compensatory weight gain was seen in the surviving females so that mean body weights were comparable with the control animals by Day 21 of lactation. The observed changes in body weight were generally accompanied by corresponding changes in food intake.
Oral exposure to the substance had no effect on oestrus cycling, fertility and mating performance or on gestation length for either generation at any dose level. Apart from the one P1 generation female at 50 mg/kg bw/day with signs of dystocia, there was no indication of difficulties during parturition in any other female. Sperm parameters were unaffected in both generations at any dose level. Sexual maturation of the P1 generation females was considered to be unaffected by exposure to the substance, with the apparent premature onset considered to be within normal biological variation. There was no effect on the primordial follicles of the P1 generation females. For both parental generations, higher group mean kidney weights were seen in males at 15 or 50 mg/kg bw/day, which was associated with an increase in hyaline droplets microscopically. Basophilia of the cortical tubules and epithelial necrosis were also seen in the kidneys at 50 mg/kg bw/day. High liver weights in males at 50 mg/kg bw/day from generations and at 5 and 15 mg/kg bw/day in the P1 generation, correlated with centrilobular hypertrophy. These effects were considered to be an adaptive response. High thyroid weights were observed in the P generation at all doses, which correlated with hypertrophy of the follicular epithelium in males at 15 and 50 mg/kg bw/day.
Microscopic changes and signs of reduced lactational activity in the mammary glands were seen in parental females given 15 or 50 mg/kg bw/day in both generations. Further changes in the female reproductive tract occurred in these females that indicated abnormal lactational dioestrus.
Maternal exposure to the substance was associated with pup mortality. The incidence of F1a pup mortality was relatively low and primarily affected the 50 mg/kg bw/day group, where total litter loss was seen for two females. Significant F2a pup mortality was observed in all dose groups, with total litter loss occurring for one, nine and 15 females given 5, 15 and 50 mg/kg bw/day. The extent of mortality at 50 mg/kg bw/day was considered to have impacted on the viability of the group and, therefore, the surviving females and litters were killed prematurely (typically on Days 3 to 6 of lactation). Pups from the affected litters were either found dead after completion of parturition or died/were euthanised in the initial days after birth (typically up to Day 2 of lactation). Clinical signs were also seen in the pups at 15 or 50 mg/kg/day in the F1a generation and at all dose levels in the F2a generation, consisting of no milk in the stomach, cold body surface and/or a flaky surface to the skin; the latter finding was most prevalent in the F1a pups, where abnormally purple/pink and/or red areas of the body were also identified. Macroscopic necropsy findings for the pups were generally limited to the absence of milk in the stomach, however, the degree of autolysis and/or cannibalisation prevented a thorough examination. Following the aforementioned pup mortality at the start of lactation, the survival indices were generally comparable with Controls from Day 4 of lactation to weaning for both generations.
Pups from the 15 or 50 mg/kg/day groups in both generations, and at 5 mg/kg/day in the F2a generation only, were significantly lighter than the Controls after birth; lower pup weights were not limited to those litters with a high incidence of pup mortality. The surviving pups gained weight similarly to Controls thereafter, however, mean body weights on Day 21 of age generally remained slightly lower.
There was no consistent effect on the anogenital distance index between the generations. The F1a pups showed a dose-related increase in mean anogenital distance on Day 1 of age, however, this finding was not replicated in F2a pups and, therefore, the toxicological significance of this finding is unclear. Nipples were not detected in the male pups from the treated groups from either generation. There were no organ weight changes or macroscopic necropsy findings in the pups from either generation that were attributed to maternal administration of the substance.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

5 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

A fully reliable GLP study conducted to OECD TG 416 is available.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

NOAEL for foetal/developmental toxicity: 10 mg/kg bw/day based on minor foetal abnormalities (e.g. oedema of the ventral surface of the body and the ventral region of the thorax) and variant abnormalities of the cervical vertebrae (rat, OECD TG 416, exposure during gestation)

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Remarks:

The study was conducted to meet the national regulatory requirements in a non-EEA country.

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start: 25 June 2020, Experimental end: 27 January 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(Han)

Details on test animals or test system and environmental conditions:

Supplier: Charles River (UK) Limited, Margate, Kent, United Kingdom
Sex: time-mated females
Age: 9 - 10 weeks of age on arrival
Acclimatisation: at least two days
Weight: 173 to 250 g
Housing: solid-floor cages (RB3 supplied by North Kent Plastics, NKP), with bedding provided. Bedding was changed regularly to maintain hygiene.
Temperature: 19 to 23 °C
Humidity: 40 to 70%
Illumination: 12 hours light to 12 hours darkness cycle
Diet: pelleted rodent diet, VRF 1 manufactured by SDS and supplied by Charles River (UK) Limited, ad libitum
Water: mains tap water, ad libitum

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

The test item was inspected for any signs of crystallisation. The required quantity of test item was added to a container, together with a magnetic stirring bar. Approximately 60% of the final required quantity of vehicle (or the total quantity of vehicle for formulations prepared by weight) was added and the mixture was stirred until the test item was suitably dispersed. For formulations prepared by volume, the formulation was made up to final quantity with the remaining vehicle and stirred again until suitably homogeneous.
Formulations were divided into daily aliquots and stored at room temperature in amber glass bottles. They were stirred from 15 minutes before the start of dosing until the completion of their use for dosing, to ensure thorough re-suspension and homogeneity, which was checked visually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were taken from all formulations. Sets of samples (for analysis or for contingency) were taken from each test item formulation prepared for the first day of dosing and were analysed for test substance using a validated method to confirm homogeneity and also achieved concentrations. Having satisfactorily confirmed achieved concentrations and homogeneity on the first day of dosing, the samples for towards the end of the dosing period were analysed simply to confirm satisfactory concentration. For Controls, sets of samples (for analysis or for contingency) were taken from the vehicle on the first day of dosing and on one occasion towards the end of the dosing period and were analysed to confirm absence of test item.
The individual measured concentrations of the test substance were within 8% of their nominal values. The analysis of formulations at the start of the study also confirmed that the formulations were homogeneous as shown by a relative standard deviation (% RSD) of 1.1% or lower. These data thereby fulfilled the acceptance criteria (±10% of nominal for achieved concentration and a % RSD no greater than 5% for homogeneity).

Details on mating procedure:

Each female had been mated with a sexually mature male of the same strain. The day on which mating was detected was designated Day 0 of gestation.

Duration of treatment / exposure:

Dosing by oral gavage from Day 6 to 19 of gestation, inclusive

Frequency of treatment:

Daily

Duration of test:

The animals were killed on Day 20 of gestation.

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

No. of animals per sex per dose:

22 females

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels were selected on the basis of results from a 14-day non-pregnant dose range-finding study, a developmental toxicity screening study (OECD TG 422) and the preliminary results of an on-going 90-day study (OECD TG 408). In the screening study, females in the 300 mg/kg/day dose group had to be euthanised by Day 9 due to general poor physical condition and body weight loss. Females given 150 mg/kg/day also showed significantly lower body weight gain in the first two weeks of the treatment period compared with the Control group. In the 90-day study, in the first half of the treatment period no test item related clinical signs or effects on the body weight parameters were observed at any dose level up to 50 mg/kg/day. Based on the female mortality and the reduced body weight gain, 100 mg/kg/day was selected as the top dose for this study. The subsequent doses of 30 mg/kg/day and 10 mg/kg/day were calculated by a factor of approximately three.
Animals were allocated to groups using a stratified body weight randomisation procedure based on individual body weights recorded on Day 0 of gestation (making sure that females mated with the same male were spread across the groups) by the supplier. The cages were positioned in the battery using a randomised cage allocation procedure, starting at the top left-hand corner of the rack and then working from left to right, top to bottom. All groups were allocated to each rack.

Maternal examinations:

Clinical observations: animals were examined twice daily for mortality and morbidity and given a detailed clinical examination daily. Animals were also observed circa one to two hours after dosing.
Body weight: the weights were recorded on Day 0 of gestation, and daily from Day 5 to 20 of gestation
Food intake: food amounts consumed by each animal were recorded over Days 6-9, 9-12, 12-15, 15-18 and 18-20 of gestation
At the termination of the studies, the animals were weighed, the thoracic and abdominal cavities were opened by a ventral mid-line incision and the major organs were examined. Gravid uterus and placenta weights were recorded and organs or tissues showing any macroscopic abnormalities were removed and retained in fixative.
The thyroids and liver from all dams were weighed after trimming of fat and other
contiguous tissue. Thyroids were weighed together after fixation.
For all dams, the liver, thyroids (including parathyroids) and any gross lesions (including abnormal teeth) were preserved in 10% buffered formalin.
For all animals, the thyroids (and abnormal teeth from dams with tooth abnormalities) were wax embedded, cut at a nominal thickness of 4 μm to 5 μm, stained with haematoxylin and eosin and examined microscopically. To aid interpretation of apparent liver weight increases, the liver from all pregnant females was wax embedded, cut at a nominal thickness of 4 μm to 5 μm, stained with haematoxylin and eosin and examined microscopically.

Ovaries and uterine content:

The uterus of any apparently non-pregnant female was stained with ammonium sulphide to confirm pregnancy status. The uterus was then retained in 70% IDA (industrial denatured alcohol) for approximately seven days and then transferred and retained in 10% buffered formalin.
For all pregnant females, the number of corpora lutea and the number and distribution of implantations in each uterine horn were recorded. Implantations were classified as early intrauterine deaths, late intrauterine deaths, dead foetuses or live foetuses.
The implantations were numbered separately for the right and left horns. Numbering was sequential, commencing at the ovarian end through to the cervix. The foetuses and their placentae were removed and the uterus and ovaries were retained in 10% buffered formalin.

Blood sampling:

Thyroid hormone assessment: Blood samples (0.2 mL) were taken on the day of necropsy from the tail vein of all animals into plain tubes (a gel separator tube without anticoagulant). All samples were taken between 08:00 and 08:42 hours and the animals were sampled in a random cage and group order. Blood samples were allowed to clot for at least 30 minutes at room temperature before being centrifuged (3000 g, 10 minutes, at approximately 4 °C). The resulting serum was aliquoted into two tubes before being stored frozen (< -70 °C) until analysis.
One aliquot of the samples was analysed for thyroxine (T4), triiodothyronine (T3) and thyroid stimulating hormone (TSH) using validated methods.

Fetal examinations:

On Day 20 of gestation, live foetuses were killed by rapid cooling, weighed, sexed and examined for external abnormalities. Anogenital distance was measured using digital callipers, measuring between the anus and caudal end of the genital tubercle.
All live foetuses were briefly placed in 70% IDA and subjected to micro-dissection, where the viscera were examined (paying particular attention to the reproductive tract for signs of altered development) and the foetuses eviscerated. Approximately 50% of the live foetuses were allocated to the fixed head examination where they were decapitated and the heads were fixed in Bouin's fluid and examined by serial sectioning. A coronal section was made through the head of the intact foetuses along the frontal parietal suture and the brain examined.
All carcasses were transferred to 95% IDA and cleared in potassium hydroxide, stained with Alizarin red S and Alcian blue to visualise the ossified skeleton and cartilage and examined.

Statistics:

Information on statistics is provided under "Any other information on materials and methods"

Indices:

Pre-implantation loss (%) is calculated as (number of corpora lutea - number of implantation sites) divided by number of corpora lutea multiplied by 100
Post-implantation loss (%) is calculated as (number of implantation sites - number of live foetuses) divided by number of implantation sites multiplied by 100
Anogenital distance index (ACGI) is calculated as the ratio of Anogenital distance (mm) to cubed root body weight (g)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 30 or 100 mg/kg/day, there was an initial, dose-related, reduction in group mean body weight gain compared with Controls, with some animals given 100 mg/kg/day losing weight over Days 6 to 9 of gestation. After this point, there was an increase in body weight, however, overall group mean body weight gain in the groups given 30 or 100 mg/kg/day was statistically significantly (p ≤ 0.01) lower than Controls. This resulted in group mean body weight on Day 20 of gestation being 5% and 6% lower than Controls, respectively.
Terminal body weight adjusted for the weight of the gravid uterus was statistically
significantly (p ≤ 0.05) lower (7%) than Controls for females given 30 or 100 mg/kg/day.
Group mean body weight, body weight gain and terminal body weight adjusted for the
weight of the gravid uterus were similar to Controls for females given 10 mg/kg/day.
However, when the terminal body weight gain was adjusted for the weight of the uterus it was 33% lower than Controls (p≤0.01).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Group mean food intake for females given 30 or 100 mg/g/day was statistically significantly (p ≤ 0.01) lower than Controls throughout the dosing period, with an overall food intake 12% and 18% lower than Controls, respectively

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At 30 or 100 mg/kg/day, there were 19% and 37%, respectively, decreases in group mean T4 concentrations (p≤0.01). The group mean T4 concentrations were outside the limited historical Control data range available for thyroid hormone concentrations on Day 20 of gestation.
Although the mean total T4 concentrations at 30 and 100 mg/kg/day were lower than those in the controls on day 20 of gestation and below the limited historical Control ranges, there were no corresponding changes to either total T3 or TSH concentrations. Given the inherent individual variability in hormone concentrations and since there were no effects on thyroid weights or microscopic changes at these dose levels, it is considered that the lowering in mean total T4 seen in this study was not adverse.

Endocrine 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 100 mg/kg/day, group mean body weight adjusted liver weights were higher than the
Controls (up to 12% higher; p≤0.01). Most individual absolute and body weight related values were outside the Control range. These changes did not correlate with the microscopic findings in the liver.
Group mean absolute liver weight was statistically significantly lower (p≤0.05 to p≤0.01) than Controls for females given 10 or 30 mg/kg/day, however, the mean adjusted liver weights and body weight-related liver weights were similar to Controls. Therefore, this slight lowering in absolute liver weight was considered not to be test item related. There was no effect on absolute or body weight adjusted thyroid weight at any dose level.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Abnormal white teeth were noted in one Control and one female given 30 mg/kg/day at
necropsy, for which there was no histopathological correlate. There were no test item-related macroscopic abnormalities noted in the liver or thyroid glands at necropsy. The observed findings were generally consistent with changes encountered in rats of this age and strain kept under laboratory conditions.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related changes were observed in the liver of females given 100 mg/kg/day. These included occasional necrotic hepatocytes (minimal) in 13 out of 22 females and an increased incidence of focal/multifocal inflammatory cell infiltrates (minimal), most of which were adjacent to the necrotic hepatocytes.
There were no microscopic findings to account for the increased liver weights in animals given 100 mg/kg/day.
Also, there were no microscopic findings to account for the abnormal white teeth noted in two females at necropsy. There were no test item-related microscopic findings in the thyroid glands examined in females given 10, 30 or 100 mg/kg/day of the test item.
Other observed findings were generally consistent with changes encountered in rats of this age and strain kept under laboratory conditions.

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

not examined

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

On Day 20 of gestation, there were 21, 20, 22 and 20 females with live foetuses in the Control group and groups given 10, 30 or 100 mg/kg/day, respectively. There was no effect on the uterine or implantation data at any dose level, with similar incidences of pre- and post-implantation loss and comparable numbers of live foetuses per litter across the groups.

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

At 30 or 100 mg/kg/day, the group mean foetal weights for male and female foetuses
combined, or separately, were slightly below the background data range, although only 3% lower than the Control group mean and there was no clear dose relationship.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

effects observed, treatment-related

Description (incidence and severity):

There was a statistically significant decrease in group mean anogenital distance index (AGDI) for male foetuses given 10, 30 and 100 mg/kg/day compared with Control (6%, 11% or 10%, respectively; p≤0.05 to p≤0.01). While this was not dose-related, the group mean values were below the limited historical Control data for all groups
given the substance.
There was a slight, dose-related increase in group mean AGDI for female foetuses compared with Control (4%, 7% or 10%, respectively) achieving statistical significance at 30 and 100 mg/kg/day (p≤0.05; p≤0.01). The group mean values were within the limited historical Control data, therefore the apparent intergroup differences were considered to reflect normal biological variation and not to be associated with treatment.

Changes in postnatal survival:

not examined

External malformations:

effects observed, treatment-related

Skeletal malformations:

effects observed, treatment-related

Visceral malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

Major foetal abnormalities were observed in five foetuses; one foetus in the Control group and in each of the groups given 30 or 100 mg/kg/day and in two foetuses from two litters in the group given 10 mg/kg/day. Details of the abnormalities were as follows:
- control group, dam 17, foetus R4: fused jugal and maxilla
- 10 mg/kg/day group, dam 30, foetus R3: malformed cervical vertebral neural arch(es)
- 10 mg/kg/day group, dam 37, foetus R6: microphthalmia
- 30 mg/kg/day group, dam 49, foetus L4: dome shaped head, cheiloschisis, cleft palate, anophthalmia, malformed premaxilla, orbital cavity reduced in size, malformed tympanic ring
- 100 mg/kg/day group, dam 75, foetus R3: incomplete interventricular septum
The nature, incidence and intergroup distribution of these major foetal abnormalities were considered to be incidental and not test item-related.

At 30 and 100 mg/kg/day, there were statistically significant higher numbers of litters with foetuses with the minor abnormality of oedema of the ventral surface of the body (p≤0.01) and the incidences were dose-related. In addition, oedema of the ventral region of the thorax was noted in foetuses from two litters in each of these groups and at 100 mg/kg/day, oedema of the ventral region of the neck was seen in foetuses from two litters and protruding tip of the tongue in foetuses from seven litters. None of these minor abnormalities has been noted in the historical Control data and were considered to be adverse.
At 100 mg/kg/day, there was a higher number of litters with foetuses with minor and variant abnormalities of the cervical vertebrae including mishapen neural arch(es), fused cartilaginous ventral plate and additional cartilaginous ventral plate, and cartilaginous additional cartilaginous ventral plate on the 5th cervical vertebra. In addition, there was an increase in the number of litters with foetuses showing the minor abnormality of nonossification of the caudal vertebral neural arches when compared with Controls. All values were outside the historical Control ranges.
Other minor and variant foetal abnormalities that attained statistical significance at 100 mg/kg/day when compared with the Controls, included partially fused jugal and maxilla, uni- or bilateral cervical rib, one or more misshapen or misaligned sternebrae, incomplete ossification of one or more metatarsals, increased pelvic cavitation, interrupted costal cartilage and non, or incomplete, ossification of the 5th sternebra. The incidences of these abnormalities were low, affecting a small number of litters and generally remained within the historical Control data ranges.
The incidences of the minor and variant foetal abnormalities that attained statistical significance when compared with the Controls are presented in a separate table.

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

external malformations

skeletal malformations

Remarks on result:

other: There was a reduction of anogenital distance in male foetuses at all dose levels

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

external: trunk

external: anogenital distance

external: thorax

skeletal: skull

skeletal: sternum

skeletal: rib

skeletal: vertebra

skeletal: hindlimb

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

30 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

presumably yes

Table 1: Minor abnormalities

Finding		Dose level (mg/kg/day)				Background data range
		0	10	30	100
Oral cavity: - protruding tongue tip	# foetuses	0	0	0	13 (6.2)JJ	Not seen
	# litters	0	0	0	7CC
Neck: - oedema in ventral region	# foetuses	0	0	0	18 (7.9)	Not seen
	# litters	0	0	0	2
Thorax: - oedema in ventral region	# foetuses	0	0	3 (5.5)	11 (5.0)J	Not seen
	# litters	0	0	2	2
Body: - oedema in ventral region	# foetuses	0	0	112 (52.6)JJ	137 (68.6)JJ	Not seen
	# litters	0	0	12CC	17CC
Skull: - jugal and maxilla: uni- or bilateral partial fusion	# foetuses	0	0	0	3 (3.1)J	1 to 4 foetuses, 0.6 to 3.1% of litters, 1 to 2 litters
	# litters	0	0	0	3C
Cervical vertebra: - one or more neural arch: misshapen	# foetuses	2 (1.0)	11 (5.4)	8 (7.5)	17 (8.9)JJ	1 to 7 foetuses, 0.5 to 4.1% of litters, 1 to 6 litters
	# litters	2	6	6	14CC
Cervical vertebra: - cartilaginous ventral plate: uni- or bilateral: fused	# foetuses	1 (0.4)	11 (6.2)	5 (2.2)	13 (6.7)JJ	1 to 4 foetuses, 0.3 to 2.5% of litters, 1 to 4 litters
	# litters	1	8	5	11CC
Cervical vertebra: - additional cartilaginous ventral plate: uni- or bilateral: fused	# foetuses	1 (0.4)	11 (6.2)	5 (2.2)	14 (7.1)JJ	1 to 4 foetuses, 0.4 to 2.5% of litters, 1 to 4 litters
	# litters	1	8	5	12CC
Caudal vertebra: - number of neural arches: 0 ossified	# foetuses	1 (0.5)	3 (1.3)	5 (2.2)	9 (4.5)J	1 to 3 foetuses, 0.4 to 3.7% of litters, 1 to 3 litters
	# litters	1	3	3	5
Rib: - uni-bilateral: cervical	# foetuses	4 (1.7)	8 (4.4)	2 (0.8)	12 (6.3)	1 to 13 foetuses, 1.0 to 12.6% of litters, 1 to 9 litters
	# litters	3	6	2	8C
Sternum: - one or more sternebra: misshapen or misaligned	# foetuses	0	1 (0.5)	5 (2.1)J	3 (1.3)J	1 to 13 foetuses, 0.7 to 5.7% of litters, 1 to 10 litters
	# litters	0	1	5	2
Hindlimb: - one or more metatarsal: incomplete ossification	# foetuses	3 (1.5)	5 (2.3)	9 (3.7)	22 (10.6)J	1 to 9 foetuses, 0.4 to 4.2% of litters, 1 to 7 litters
	# litters	3	4	5	9C

Figures in brackets are group mean percentage values; J = p<0.05, JJ = p<0.01 (Jonckheere Trend Test); C = p<0.05, CC = p<0.01 (Cochran-Armitage Test)

Table 2: Variations

Finding		Dose level (mg/kg/day)				Background data range
		0	10	30	100
Abdominal cavity: - kidney, uni- or bilateral: increased pelvic cavitation	# foetuses	0	0	1 (0.4)	3 (1.7)J	1 to 5 foetuses, 0.5 to 5.4% of litters, 1 to 3 litters
	# litters	0	0	1	2
Cervical vertebra: - ventral tubercle: not ossified	# foetuses	63 (31.3)	102 (45.3)J	113 (51.2)JJ	131 (61.6)JJ	26 to 107 foetuses, 24.3 to 52.4% of litters, 12 to 21 litters
	# litters	18	19	21	20C
Cervical vertebra: - cartilaginous ventral plate: uni- or bilateral : on 5th cervical vertebra	# foetuses	2 (1.0)	10 (4.9)	7 (6.9)	15 (8.0)JJ	1 to 9 foetuses, 0.4 to 5.9% of litters, 1 to 6 litters
	# litters	2	6	5	13CC
Cervical vertebra: - additional cart ventral plate: uni- or bilateral: on 5th cervical vertebra	# foetuses	1 (0.4)	11 (6.2)	4 (1.6)	17 (8.5)JJ	1 to 6 foetuses, 0.3 to 2.7% of litters, 1 to 5 litters
	# litters	1	8	4	14CC
Rib: - one or more: costal cartilage interrupted	# foetuses	75 (36.8)	86 (40.7)	116 (52.0)J	101 (49.6)J	1 to 108 foetuses, 0.7 to 50.5% of litters, 1 to 22 litters
	# litters	20	20	22	20
Sternum: - 5th sternebra: not ossified	# foetuses	7 (3.4)	13 (6.0)	11 (5.0)	33 (15.8)JJ	1 to 13 foetuses, 0.4 to 10.7% of litters, 1 to 7 litters
	# litters	3	9	6	12CC
Sternum: - 5th sternebra: incomplete ossification	# foetuses	18 (8.8)	22 (9.9)	36 (15.0)	42 (19.4)J	1 to 20 foetuses, 1 to 15.3% of litters, 1 to 11 litters
	# litters	11	10	15	15C
Forelimb: - one or more digits – bilateral: phalanges ossified	# foetuses	29 (12.2)	40 (18.6)	49 (26.0)	56 (29.1)J	15 to 75 foetuses, 9.4 to 45.4% of litters 9 to 15 litters
	# litters	9	7	15	12

Figures in brackets are group mean percentage values; J = p<0.05, JJ = p<0.01 (Jonckheere Trend Test); C = p<0.05, CC = p<0.01 (Cochran-Armitage Test)

Conclusions:

The NOAEL for maternal toxicity and embryo-foetal development was considered to be 10 mg/kg/day.

Executive summary:

The developmental toxicity of the substance was studied under GLP to OECD TG 414. Four groups of 22 sexually mature, time-mated female Crl:WI(Han) rats were given the test substance in corn oil by gavage at zero (vehicle control), 10, 30 or 100 mg/kg bw/day, once daily, at a dose volume of 2 mL/kg from Day 16 to 19 of gestation, inclusive. There were no premature deaths and no abnormal clinical signs of maternal toxicity. There was a dose-related reduction in maternal body weight and food consumption at 30 or 100 mg/kg bw/day. At these doses, there was a statistically significant decrease in group mean T4 concentrations (19% and 37%, respectively) compared to the control animals. However, there was no effect on T3 and TSH concentrations at 30 or 100 mg/kg bw/day. Furthermore, there was no effect on thyroid weights or microscopic findings, and thus the decreased T4 concentrations were considered to be not adverse. At 100 mg/kg bw/day only, group mean absolute and body weight-related liver weights were up to 12% higher than that of the controls. Macroscopic findings of abnormal white teeth were noted in one control and in one female given 30 mg/kg bw/day, for which there was no histopathological correlate. Substance-related microscopic minimal changes of occasional necrotic hepatocytes were observed in the liver of 13 out of 22 females given 100 mg/kg bw/day. Furthermore, there was an increased incidence of focal/multifocal inflammatory cell infiltrate (minimal) at this dose level. On day 20 of gestation, there were 21, 20, 22 and 20 females with live foetuses in the control group and the groups given 10, 30 or 100 mg/kg bw/day, respectively. There was no effect on the uterine or implantation data, foetal weight, placental weight or the mean sex ratio at any dose level that was considered to be associated with the test substance. The group mean anogenital distance index (AGDI) for male foetuses at 10, 30 and 1000 mg/kg bw/day was significantly lower than that of controls (6%, 11% or 10%, respectively). There were no major foetal abnormalities that were attributed to the test substance. At 30 or 100 mg/kg bw/day there was a higher number of litters with foetuses that had the minor foetal abnormality of oedema. Furthermore, at 100 mg/kg bw/day there was a higher number of litters with foetuses with a protruding tip of the tongue and minor or variant abnormalities of the cervical vertebrae. 

In conclusion, the administration of the substance to pregnant Crl:WI(Han) rats once daily by oral gavage from days 6 to 19 of gestation, inclusive, was well tolerated at dose levels of 10, 30 or 100 mg/kg bw/day. Signs of maternal toxicity were limited to 30 or 100 mg/kg bw/day. There was no effect of the substance on pregnancy or foetal growth. However, there was an increase in the incidences of minor foetal abnormalities at 30 or 100 mg/kg bw/day. Based on the above findings, the NOAEL for maternal toxicity and embryo-foetal development was considered to be 10 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

10 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

A fully reliable GLP study conducted to OECD TG 414 is available.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Toxicity to reproduction: other studies

Additional information

The substance has been evaluated for its potential to cause effects on reproductive and developmental toxicity in a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test, a two-generation reproductive toxicity study and in a prenatal developmental toxicity study, all in the rat. In all studies, there was no evidence to suggest effects of the substance on sexual function or fertility in rats.

Reproduction/developmental toxicity screening study
In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD TG 422), four groups of 10 male and 10 female Crl:WI(Han) rats were given 0 (vehicle – corn oil), 30, 150 or 300 mg/kg/day of the test material by oral gavage. Males were dosed for approximately 8 weeks from 14 days before and during pairing and until the day before necropsy. Due to excessive body weight loss and a decline in clinical condition, females given 300 mg/kg/day were only dosed for 8 days before being sent to necropsy; the remaining females were dosed for 14 days before pairing, during pairing and gestation and until Day 12 of lactation. A NOAEL for systemic toxicity could not be established based on histopathological changes at 30 mg/kg/day in the teeth (atrophy of ameloblasts) in females and lower body weight gain and histopathological changes in the liver (diffuse or centrilobular hypertrophy), thyroid gland (epithelial hypertrophy accompanied by increased basophilia of the colloid) and forestomach (epithelial hyperplasia with associated hyperkeratosis) in males. There was no effect on oestrous cycling, fertility and mating performance, or on gestation and parturition and therefore the NOAEL for reproductive performance was considered to be 150 mg/kg/day. A NOAEL for developmental toxicity could not be established based on a slight, but dose-related reduction in the number of pups born alive and lower pup survival from Day 1 to 4 of age, with lower pup weight gain and T4 concentrations in the surviving offspring.

2-generation reproductive toxicity study
In a subsequent two-generation reproduction study (OECD TG 416), the substance was administered to Crl:WI(Han) rats by oral gavage at 0 (vehicle – corn oil) 5, 15 or 50 mg/kg/day for at least 10 weeks before mating, during mating, gestation and lactation, throughout two generations. Maternal administration of the substance was associated with pup mortality at all dose levels. During the P generation, the incidence of F1a pup mortality was relatively low and primarily affected the 50 mg/kg/day group, where total litter loss was seen for two females only. During the F1 generation, however, significant F2a pup mortality was seen at all dose levels of the substance, with total litter loss for 1, 9 and 15 females given 5, 15 and 50 mg/kg/day, respectively; the extent of mortality at 50 mg/kg/day was considered to have impacted on the viability of the group and therefore, the surviving females and litters were euthanised prematurely. Pups from the affected litters were either found dead after completion of parturition or died/were euthanised in the initial days after birth. Although there were no effects on reproductive performance, mating behaviour or conception, the effects on pup survival and reduced birth weight at all dose levels in the second generation prevented a No Observed Adverse Effect Level (NOAEL) from being established for effects on reproduction. Signs of systemic toxicity in the adult animals were characterised by adaptive liver hypertrophy in both generations and hypertrophy of the thyroid follicular epithelium during the parental generation only. Changes in the kidney were also seen in males from both generations given 15 or 50 mg/kg/day, as characterised by increased hyaline droplets and basophilia, with epithelial necrosis/distension of the cortico-medullary tubules. Although the kidney findings may be indicative of rodent specific effects, they were considered to be adverse in the context of this study. Therefore, the NOAEL for systemic toxicity was defined as 5 mg/kg/day.

Pre-natal developmental toxicity study
The substance has been tested for developmental toxicity in Crl:WI(Han) rats (OECD TG 414), where administration to pregnant rats once daily by oral gavage from Days 6 to 19 of gestation, inclusive, at dose levels of 0 (vehicle – corn oil), 10, 30 or 100 mg/kg/day was well tolerated. Signs of maternal toxicity were limited to 30 or 100 mg/kg/day, where there was a reduction in body weight gain and food intake and decreased thyroid hormone concentrations (T4). Increased liver weight and microscopic findings in the liver were seen at 100 mg/kg/day. There was no effect of the substance on pregnancy or foetal growth, however, there was a reduction of anogenital distance in male foetuses at all dose levels. At 30 or 100 mg/kg/day, there were increases in the incidences of minor foetal abnormalities, including oedema of the ventral surface of the body and oedema of the ventral region of the thorax. At 100 mg/kg/day, oedema of the ventral region of the neck was also observed as well as a higher number of litters with foetuses with minor and variant abnormalities of the cervical vertebrae including mishappen neural arch(es), fused cartilaginous ventral plate and additional cartilaginous ventral plate, and additional cartilaginous ventral plate on the 5th cervical vertebra. In addition, there was an increase in the number of litters with foetuses showing the minor abnormality of non-ossification of the caudal vertebral neural arches. All values were outside the historical control ranges. Based on the above findings, the No Observed Adverse Effect Level (NOAEL) for maternal toxicity and embryo-foetal development was considered to be 10 mg/kg/day.

Justification for classification or non-classification

Developmental toxicity (significant decrease in the number of live pups at birth, decreased pup weight after birth and increased pup mortality and total litter losses) was clearly observed in the two-generation study in rats, with more severe effects and all dose levels affected in the F1 generation litters (F2a pups). Lower pup survival was also seen in the reproduction/developmental toxicity screening test at all dose levels. There were no indications that the increased pup mortality was secondary to maternal toxicity or was caused via lactation and therefore the substance meets the criteria for classification as toxic for reproduction Category 1B: May damage the unborn child (H360D) under Regulation 1272/2008.