2-{2-chloro-4-mesyl-3-[(tetrahydrofuran-2-ylmethoxy)methyl]benzoyl}cyclohexane-1,3-dione

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Two-Generation Toxicity Study (OECD 416), rats:

NOAEL systemic = 2 ppm corresponding to:

NOAEL systemic (males, P1) = 0.126 mg/kg bw/day

NOAEL systemic (females, P1) = 0.202 mg/kg bw/day

NOAEL systemic (males, F1) = 0.142 mg/kg bw/day

NOAEL systemic (females, F1) = 0.204 mg/kg bw/day

NOAEL fertility >= 200 ppm (highest dose tested) corresponding to:

NOAEL fertility (males, P1) >= 13.1 mg/kg bw/day

NOAEL fertility (females, P1) >= 20.4 mg/kg bw/day

NOAEL fertility (males, F1) >= 14.6 mg/kg bw/day

NOAEL fertility (females, F1) >= 20.9 mg/kg bw/day

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Aug 2005 - 9 Nov 2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

Results tables are missing in the translated version of the study report.

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

adopted Jan 2001

Deviations:

yes

Remarks:

results tables are missing in the translated study report

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: BrlHan:WIST@Jcl[GALAS]

Details on species / strain selection:

The Wistar Hannover GALAS rat was selected because the rat is a commonly used mammalian species for reproduction toxicity studies of agricultural chemicals and because this strain of rats yields stable reproduction results.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Fuji Breeding Center, CLEA Japan, Inc., Shizuoka, Japan
- Age at study initiation: 4 - 5 weeks
- Weight at study initiation: 116 - 133 g (males), 89 - 103 g (females)
- Housing: Pre-mating: groups of 3 animals per sex per cage in wire-mesh stainless steel cages, Mating: 1:1 in an aluminum cage with wire-mesh floor and front and a subfloor tray (W260 x D400 X H240 mm), Gestation and lactation: individually in aluminum breeding boxes (W260 x D400 x H200 mm) with nesting materials (Sunflake: Oriental Yeast Co., Ltd., Tokyo, Japan), after weaning, offspring were housed by litter in the breeding boxes; males that accomplished mating and females that accomplished lactation were housed until necropsy in wire-mesh stainless steel cages
- Diet: certified pulverized feed (MF Mash, Oriental Yeast Co., Ltd., Tokyo, Japan), ad libitum
- Water: well water purified with a rapid filtration-activated carbon adsorption system and sterilized with sodium hypochlorite, ad libitum
- Acclimation period: 6 days

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

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: 10 times during the study, once in one to 6 weeks
- Storage temperature of food: The prepared test diets were sealed in plastic bags and stored in a dark and cold room (1 - 10 °C)

Details on mating procedure:

- M/F ratio per cage: 1:1
- Proof of pregnancy: presence of vaginal plug / sperm in vaginal smears referred to as Day 0 of pregnancy
- After successful mating each pregnant female was caged: individually in aluminum breeding boxes (W260 x D400 x H200 mm) with nesting materials

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

In the dose range-finding study previously performed (IET 04-0510, 2006), it was confirmed that the test substance was stable in the basal feed at concentrations of 2 and 2000 ppm for 56 days after preparation (5 weeks in a cold and dark room with a tightly-packed condition, and 21 days in an animal room).
Chemical analyses for homogeneity and concentration of the test substance in the basal feed were performed at the first and 6th preparation samples taken from the top, middle, and bottom portions of the mixer for each dose level. Storage conditions of test diets were monitored at the 3rd and 8th preparations. Samples taken from the middle portion of the mixer immediately after preparation and from the last batch of the preparations after use were analyzed for the concentration of test substance in the basal feed at each dose level. Control diets were also analyzed in the same manner to ensure that the basal feed was not contaminated by the test substance. All scheduled analyses at the diet preparation were performed before the first use of the prepared diets except for monitoring storage condition. In the homogeneity study, the coefficient of variation of mean test substance concentration in the diet for each dose level ranged 3.0 - 7.9%, being confirmed a homogeneous distribution of the test substance in basal feed. In the concentration analyses of test diets in each dose group, the concentration of test substance in the diet at dose level of 2 ppm prepared at the 8th preparation were slightly low, 80% to the target concentration. However, in the analyses of the final batch in the same lot, an analytical value of the sample at dose level of 2 ppm was 95% to the target concentration, being concluded no problem in the test diet and storage condition of that lot. In other concentration analyses just after the preparation, the concentration of test diets in each dose group ranged 95 - 107% to the target concentrations, indicating the good condition of preparation. Furthermore, in the analysis of the final batches divided in each dose group, all analytical values ranged 85 - 104% to the target concentrations, being considered no problem in the storage condition of test diets prepared.

Duration of treatment / exposure:

approximately 18 weeks (P and F1 generations)

Frequency of treatment:

continuously (via diet)

Details on study schedule:

- F1 parental animals not mated until 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 - 25 days of age

Dose / conc.:

2 ppm

Remarks:

equivalent to 0.126, 0.202, 0.142 and 0.204 mg/kg bw/day in (P) males and females and (F1) males and females, respectively

Dose / conc.:

20 ppm

Remarks:

equivalent to 1.25, 2.03, 1.40 and 2.03 mg/kg bw/day in (P) males and females and (F1) males and females, respectively

Dose / conc.:

200 ppm

Remarks:

equivalent to 13.1, 20.4, 14.6 and 20.9 mg/kg bw/day in (P) males and females and (F1) males and females, respectively

No. of animals per sex per dose:

24

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dose levels were selected based on the results of a reproduction toxicity dose range-finding study in rats (report no. IET 04-0031, 2006). In the study, the test substance was administered to 8 SPF Wistar Hannover (BrlHan:WIST@Jcl[GALAS]) rats per sex per group at dietary concentrations of 2, 20, 200, and 2000 ppm for a period of 3 weeks prior to mating and until weaning of their F1 offspring. In the results, the body weights of male and female parental animals at 2000 ppm decreased statistically significantly and the body weight of male parental animals at 200 ppm also decreased slightly with no statistical significance. In the offspring, the body weights of males at 200 ppm and higher dose groups and females at 2000 ppm decreased statistically significantly. In addition, both parental animals and offspring showed opacity of the eyeball, and the finding which was the most sensitive in female parental animals were noted in 2, 6, and 6 animals at 20, 200, and 2000 ppm, respectively. On the other hand, increases in the liver weights and other findings were noted in both the parental animals and their offspring, whereas no remarkable toxicological effects in any items suspected in relation with the treatment of test substance were noted in the parental animals and their offspring at 2 ppm. Based on the results, a dose level of 2 ppm was selected as the low-dose level, and dose levels of 20 ppm and 200 ppm were selected as the middle-dose and high-dose levels, respectively.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (once on holidays)
- Cage side observations included: mortality/viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Parental animals of both sexes were observed from cage-side twice daily (once on holidays) for clinical signs. Each animal was also examined in detail for the presence of abnormalities such as excitement, convulsion, sedation, and abnormal gait when it was weighed. During breeding, females were examined for the status of pregnancy and parturition. Any abnormalities were recorded along with the date of onset, nature, degree, and duration.

BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed on the day of initiation of dosing, weekly during the pre-mating growth and breeding periods and on the day of necropsy.
Females were weighed on the day of initiation of dosing, weekly during the pre-mating growth period, on Days 0, 7, 14, and 20 of gestation and on Days 0, 7, 14, and 21 of lactation during the breeding period and on the day of necropsy. These measurements were made on females that lost all pups during the lactation period. Females not producing offspring were weighed weekly on Day 25 and thereafter of post coitus and on the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Food consumption of lactating females was expressed as the total amounts of food consumed by maternal animals and their offspring. During the mating period, food consumption was not determined for either sex.
- Compound intake calculated as time-weighted averages from the consumption and body weight data: Yes

WATER CONSUMPTION: No

Oestrous cyclicity (parental animals):

Vaginal smears were taken from females in each group, stained with Giemsa solution, and examined microscopically. Regularity in microscopic changes of vaginal smears associated with various stages of the oestrous cycle in females was checked for more than 2 weeks. Females periodically showing oestrous vaginal smears were considered to be normal and the percentage of normal females was calculated for each group. Oestrous cycle length of each female was expressed as mean number of days from the day of oestrous to the day before next estrus. Mean oestrous cycle length was calculated for each group. When oestrous continued 2 days, calculation of oestrous cycle length was based on the first day of oestrous.

Sperm parameters (parental animals):

Parameters examined in all P and F1 male parental generations:
At necropsy, sperm heads and sperm were collected from the testis and cauda epididymis (right side in principle), respectively, of all (P) and (F1) parental males. The number of testicular sperm heads was enumerated by using a hemacytometer. The number and motility of cauda epididymal sperm were examined by a computer-assisted sperm analysis system. Morphology of cauda epididymal sperm was performed microscopically after fixation in 10% neutral-buffered formalin.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 8 pups/litter (4/sex/litter as nearly as possible)

PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring: still births, live births, postnatal mortality, clinical signs, complete external examinations, body weight, number and sex of pups. The number of pups surviving in each litter was counted on Days 0, 4, 7, 14, and 21 of lactation. In each litter, pups were weighed on Days 0, 4, 7, 14, and 21 of lactation. Body weight measurement was performed by sex on lactation day 0 and individually on lactation day 4 and thereafter. Then mean body weights were calculated for each sex for each group.

GROSS EXAMINATION OF DEAD PUPS:
yes, for gross findings

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: all surviving animals after weaning of F1 pups
- Maternal animals: all surviving animals after weaning of F1 pups

GROSS NECROPSY
- Gross necropsy consisted of external examinations. Parental females were examined through vaginal smears for oestrous cycle stage before necropsy and the animals showing metoestrus or dioestrus were necropsied in order because the effects of oestrous cycle stage on the changes of uterus weight were reduced as small as possible.

HISTOPATHOLOGY / ORGAN WEIGHTS
After necropsy, the following organs from all P and F1 parental animals were weighed: brain, thyroid, pituitary, liver, kidneys, adrenals, spleen, ovaries, uterus (with cervix and oviducts), testes, epididymides, seminal vesicles with fluids (including coagulating glands), and prostate (ventral lobe).

The eyeballs (both sides), vagina and organs with gross abnormalities as well as the organs weighed were fixed and preserved in 10% neutral-buffered formalin with the exception of the testes which were fixed in a FSA (formalin-sucrose-acetic acid) solution.

Histopathology of the following organs was performed for 10 pairs of P and F1 parental animals selected randomly from each in the control and high dose groups, that accomplished the rearing of offspring until weaning: reproductive organs (ovaries, oviducts, uterus (horns and cervix), vagina, testis (left side), epididymis (left side), seminal vesicles, coagulating glands, and prostate), pituitary, and adrenals. The testis of all P males was examined histopathologically because the relative weights of the testes in all treated groups increased significantly in P generation. In addition, the epididymis of remaining P males in the control and high-dose groups was examined histopathologically because the relative weight of the epididymides in each treated group increased. In these histopathological examinations, abnormalities of spermatogenesis in the testis were examined in detail. The examination of the epididymis was performed on its head, body, and cauda. The ovaries of F1 females were examined for the number of primordial follicles.
Histopathology of the reproductive organs, pituitary and adrenal was carried out in pairs of animals that failed to mate or pregnancy and in maternal females that showed abnormal parturition or whole litter losses. In addition, the eyeballs (both sides) and liver in all P and F1 parental animals were examined histopathologically because these organs were considered to be the target organs.
On the other hand, the kidneys and thyroids in male parental animals were examined histopathologically because weights of these organs increased in relation with the treatment of test substance. The kidneys in the control and high-dose groups were examined in 24 animals of each group in P generation and 10 animals of each group in F1 generation. The thyroids were examined histopathologically in all P and F1 parental male animals. Furthermore, histopathology of the organs showing gross abnormalities at necropsy was performed when the study director deemed it necessary.

Postmortem examinations (offspring):

SACRIFICE
- The F1 and F2 offspring not selected on Day 4 of lactation were sacrificed and subjected to necropsy.
- The F1 offspring not selected as parental animals and all F2 weanlings were sacrificed at 26 days of age and gross findings were recorded.

GROSS NECROPSY
Of F1 and F2 weanlings in each litter in each group, one male and one female that were ranked first by sex were subjected to body weight and organ weight measurements on the day of necropsy. Organ weight measurement was performed on the brain, spleen, thymus, liver, and uterus.
The measured organs were fixed and preserved in 10% neutral-buffered formalin along with the reproductive organs, eyeballs (both sides), and organs showing gross abnormalities.

HISTOPATHOLOGY / ORGAN WEIGTHS
The eyeballs (both sides) and liver of all F1 and F2 weanlings whose organs were weighed were subjected to histopathology.

Statistics:

Please see entry under "Any other information on materials and methods incl. tables".

Reproductive indices:

Male mating index (%) = (number of copulations / number of males used for mating) x 100
Female mating index (%) = (number of copulations / number of females used for mating) x 100
Fertility index (%) = (number of pregnancies / number of females copulated) x 100
Gestation index (%) = (number of normal parturitions / number of pregnancies) x 100
Duration of gestation = days from detection of copulation to completion of parturition
Number of implantation sites = number of implantation sites in the uterus

Offspring viability indices:

Viability index on lactation day 0 (%) = (number of pups alive on lactation day 0 / number of pups delivered) x 100
Viability index on lactation day 4 (%) = (number of pups alive on lactation day 4 / number of pups alive on lactation day 0) x 100
Viability index on lactation day 7 (%) = (number of pups alive on lactation day 7 / number of pups selected on lactation day 4) x 100
Viability index on lactation day 14 (%) = (number of pups alive on lactation day 14 / number of pups selected on lactation day 4) x 100
Viability index on lactation day 21 (weaning index, %) = (number of pups alive on lactation day 21 / number of pups selected on lactation day 4) x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In clinical observation, opacity of the eyeballs and coarsened corneal surface were noted in males and females at 20 and 200 ppm. In addition, swelling of the mammary gland was noted in one to 3 females at 20 and 200 ppm just after weaning. On the other hand, no findings were noted in the eyeballs and mammary gland at 2 ppm.
At 200 ppm, the incidence of loss of fur in females was statistically significantly higher than that in the control group. However, the incidences of loss of fur in F1 females in each treated group were not significantly different from those in the control group, concluded that the significant increase of this incidence would be unrelated to the treatment of test substance.
Furthermore, eye discharge, supernumerary incisor and wound were noted in one to 2 males and females in each group. However, there were no statistical significant differences of those incidences between the treatment groups and the control group, being not suspected in relation with the treatment of test substance.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

Not applicable.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weights and body weight gain of males and females at 2 and 20 ppm group were similar to those in the control group.
At 200 ppm, the body weights were reduced in males and females reaching statistically significance in males at Week 17 of treatment and in females at Week 8 of treatment, on GD 14 and on Day 7 and 14 of lactation. The body weight gains were slightly reduced in males from Week 0 until 17 of treatment and significantly in females at Week 3 and thereafter in the growth period and on Days 0-7 and 0-14 of lactation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The food consumptions of males in each treated group were similar to those in the control group throughout the treatment period except for accidental significant high values at 2 or 200 ppm from Week 1 to 2 of treatment. Significant reduced food consumption was observed in females at 20 and 200 ppm on Days 0-7 of lactation and during the lactation period, respectively.

Food efficiency:

not examined

Description (incidence and severity):

Not applicable.

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

not examined

Description (incidence and severity):

Not applicable.

Ophthalmological findings:

not examined

Description (incidence and severity):

Not applicable.

Haematological findings:

not examined

Description (incidence and severity):

Not applicable.

Clinical biochemistry findings:

not examined

Description (incidence and severity):

Not applicable.

Endocrine findings:

not examined

Description (incidence and severity):

Not applicable.

Urinalysis findings:

not examined

Description (incidence and severity):

Not applicable.

Behaviour (functional findings):

not examined

Description (incidence and severity):

Not applicable.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Not applicable.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The liver and eyeballs in all animals were examined histopathologically because those organs were considered to be a common target of the test substance in both sexes. In the liver, centrilobular hypertrophy of the hepatocytes was observed in males at 20 and 200 ppm and the incidences were statistically significantly higher than those in the control group. However, this finding was not observed in males at 2 ppm and females in all treated groups. In addition, single cell necrosis or focal necrosis of the hepatocytes, microgranuloma and hepatodiaphragmatic nodule were observed in males and females in each treated group including the control group and those findings occurred singly in each treated group. In the eyeballs, opacity and coarsened corneal surface noted grossly were diagnosed histopathologically as keratitis. In addition, the incidences of the findings in males and females at 20 and 200 ppm groups were statistically significantly higher than those in the control group. On the other hand, no keratitis was observed in any males and females at 2 ppm. In the eyeballs, furthermore, calcification in the cornea was observed in each treated group including the control group, whereas the incidence in each group was one or 2 animals per group. Additionally, the thyroids, kidneys, testes, epididymides and seminal vesicle with coagulating gland with suspected relations with the treatment of test substance and weight gain were examined histopathologically. In the thyroid, colloidal alteration, hypertrophy of the follicular cell and increased large-sized follicles were observed in each treated group including the control group and the statistically significant differences in these incidences were occasionally noted at 20 and 200 ppm. Hydropic degeneration of the follicular cell, which has been known as one of spontaneously occurring lesions in this strain of rats, was also observed in one to 3 animals in each group, whereas there were no significant differences of the incidences between each treated group and the control group. In the kidneys, there were no findings suspected the relation with the treatment of test substance in all animals at 200 ppm. The testes, epididymides and seminal vesicle with coagulating gland were examined in all animals selected. In results, atrophy of the seminiferous tubule was observed in one to 3 animals in each group including the control group, whereas there were no abnormal findings in relation with the cause of increased organ weight.
In addition to the organs described above, the reproductive organs, pituitary and adrenals were histopathologically examined in each 10 males and females selected from the control and high-dose groups. However, there were no findings suspected in relation with the treatment of test substance in these organs. On the other hand, other gross lesions were examined histopathologically as necessary, whereas there were no abnormal findings in relation with the treatment of test substance.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

In sperm examination, sperm head count in the testis, sperm counts in the cauda epididymis and sperm motility in each group were not statistically significantly different from those in the control group. Statistically significant low incidences of sperm with normal morphology were noted at 20 and 200 ppm. However, these values were 97.9 and 98.1%, respectively, and ranged within the background data (97.4-99.2%), being considered to be of no toxicological significance.

Reproductive performance:

no effects observed

Description (incidence and severity):

No significant differences of mating index between each treated group and the control group, and mating was noted in all pairs. There were also no statistically significant differences of the number of days until mating and mating was completed in almost all animals for 1 day or 2 days after pairing. On the other hand, 6 pairs were completed to mate for 3 and more days, whereas those pairs belonged to each treated group including the control group at a low incidence such as one or less in each group.
Fertility indices of females in each treated group including the control group were favorable ranging from 87.5 to 100.0% and there were no statistically significant differences between each treated group and the control group. The numbers of females without gestation were one female at 20 and 200 ppm, respectively.
Gestation indices in each treated group including the control group were 100%.
Durations of gestation in females in each treated group were similar to those in the control group and almost all females having live pups delivered at Day 22 of gestation. Other remaining females delivered at Day 21 or 23 of gestation.
Numbers of implantation sites in females in each treated group were similar to those in the control group.
The mean numbers of pups delivered in each treated group were similar to those in the control group.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

2 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effect observed at this dose level

Remarks on result:

other: equivalent to 0.126 and 0.202 mg/kg bw/day in males and females, respectively

Key result

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

20 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Remarks on result:

other: equivalent to 1.25 and 2.03 mg/kg bw/day in males and females, respectively

Key result

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

>= 200 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: not determinable due to absence of adverse toxic effects

Remarks on result:

other: equivalent to 13.1 and 20.4 mg/kg bw/day in males and females, respectively

Critical effects observed:

yes

Lowest effective dose / conc.:

20 ppm

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Critical effects observed:

yes

Lowest effective dose / conc.:

20 ppm

System:

endocrine system

Organ:

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Almost all animals at 200 ppm showed either opacity of the eyeballs or coarsened corneal surface.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

Not applicable.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weights of parental males and females at 2 ppm were similar to those in the control group.
At 20 ppm, the body weight and body weight gain of F1 parental males was slightly lower without statistical significance.
At 200 ppm, the body weights were reduced in males and females reaching statistically significance in males throughout the treatment period and in females from the initiation of treatment to Week 4 of treatment. The body weight gains were significantly reduced in males during the treatment period and in females on Days 0-7 and 0-14 of lactation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The food consumption was significantly reduced in males at 20 ppm in Week 14 and thereafter of treatment and at 200 ppm in Week 1, Week 4-7 and Week 13 and thereafter of treatment, respectively.

Food efficiency:

not examined

Description (incidence and severity):

Not applicable.

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

not examined

Description (incidence and severity):

Not applicable.

Ophthalmological findings:

not examined

Description (incidence and severity):

Not applicable.

Haematological findings:

not examined

Description (incidence and severity):

Not applicable.

Clinical biochemistry findings:

not examined

Description (incidence and severity):

Not applicable.

Endocrine findings:

not examined

Description (incidence and severity):

Not applicable.

Urinalysis findings:

not examined

Description (incidence and severity):

Not applicable.

Behaviour (functional findings):

not examined

Description (incidence and severity):

Not applicable.

Immunological findings:

not examined

Description (incidence and severity):

Not applicable.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In males in each treated group, statistically significant differences of the absolute and/or relative weights of the organs except for the prostate were noted occasionally between each treated groups and the control group. In these changes, the relative weights of the liver, kidneys and adrenals increased significantly at 20 and 200 ppm. Those changes were related to the dose levels, being considered to be related to the treatment of test substance. There were no statistically significant differences of these organ weights observed at 2 ppm. Otherwise, the relative weights of the testes at 20 and 200 ppm increased significantly, being suspected to be related to the treatment of test substance. In addition, the absolute weights of the epididymides and seminal vesicles at 20 and 200 ppm and were slightly higher than those in the control group. The relative weights of the seminal vesicles at 200 ppm reached statistically significance. Changes of the organ weights in females were similar to those in males. The absolute weights of the liver at 2 and 20 ppm and the relative weights at 20 and 200 ppm were statistically significantly different from those in the control group. In addition, the adrenal weights at 20 and 200 ppm were increased slightly. The absolute weights at 20 ppm and the relative weights at 20 and 200 ppm were statistically significantly different from those in the control group, respectively. Otherwise, the absolute weights of the brain at 20 and 200 ppm were significantly decreased and the relative weights at 200 ppm.
No statistically significant differences of other organ weights were observed between each treated group and the control group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At 20 and 200 ppm, opacity and coarsened corneal surfaces of the eyeballs were frequently noted in males and females, either or both incidences of those findings were statistically significantly higher than those in the control group. On the other hand, enlargement of the thyroid, hepatodiaphragmatic nodule, coarse surface and pelvic dilatation of the kidneys, luminal dilatation of the common bile duct, and others were noted in each group including the control group, whereas there were no statistically significant differences of the incidences of these findings between each treated and control group.

Neuropathological findings:

not examined

Description (incidence and severity):

Not applicable.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The liver and eyeballs in all animals were examined histopathologically because those organs were considered to be a common target of the test substance in both sexes. In the liver, centrilobular hypertrophy of the hepatocytes was observed in males at 20 and 200 ppm and the incidences were statistically significantly higher than those in the control group. However, this finding was not observed in males at 2 ppm and females in all treated groups. In addition, single cell necrosis or focal necrosis of the hepatocytes, microgranuloma and hepatodiaphragmatic nodule were observed in males and females in each treated group including the control group and those findings occurred singly in each treated group. In the eyeballs, opacity and coarsened corneal surface noted grossly were diagnosed histopathologically as keratitis. In addition, the incidences of the findings in males and females at 20 and 200 ppm groups were statistically significantly higher than those in the control group. On the other hand, no keratitis was observed in any males and females at 2 ppm. In the eyeballs, furthermore, calcification in the cornea was observed in each treated group including the control group, whereas the incidence in each group was one or 2 animals per group. Additionally, the thyroids, kidneys, testes, epididymides and seminal vesicle with coagulating gland with suspected relations with the treatment of test substance and weight gain were examined histopathologically. In the thyroid, colloidal alteration, hypertrophy of the follicular cell and increased large-sized follicles were observed in each treated group including the control group and the statistically significant differences in these incidences were occasionally noted at 20 and 200 ppm. Hydropic degeneration of the follicular cell, which has been known as one of spontaneously occurring lesions in this strain of rats, was also observed in one to 3 animals in each group, whereas there were no significant differences of the incidences between each treated group and the control group. Chronic nephropathy was observed in two (one of them showing coarse surface) of 12 animals (10 selected animals and two animals with gross findings) at 200 ppm. The testes, epididymides and seminal vesicle with coagulating gland were examined in all animals selected. In results, atrophy of the seminiferous tubule was observed in one to 3 animals in each group including the control group, whereas there were no abnormal findings in relation with the cause of increased organ weight.
In females at 200 ppm, the number of primordial follicles was similar to that in the control group. The mammary gland which was grossly hypertrophic was confirmed histopathologically as incomplete recovery from lactation phase, whereas there were no findings indicating toxicological effects.
In addition to the organs described above, the reproductive organs, pituitary and adrenals were histopathologically examined in each 10 males and females selected from the control and high-dose groups. However, there were no findings suspected in relation with the treatment of test substance in these organs. On the other hand, other gross lesions were examined histopathologically as necessary, whereas there were no abnormal findings in relation with the treatment of test substance.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

In sperm examination, sperm head count in the testis, sperm counts in the cauda epididymis and sperm motility in each group were not statistically significantly different from those in the control group.

Reproductive performance:

no effects observed

Description (incidence and severity):

No significant differences of mating index between each treated group and the control group, and mating was noted in all pairs except for one pair at 200 ppm. Mating of this pair was not completed until 2 weeks after pairing, whereas mating was completed by exchanging the male by another which was already successful in mating. There were also no statistically significant differences of the number of days until mating and mating was completed in almost all animals for 1 day or 2 days after pairing. On the other hand, 6 pairs were completed to mate for 3 and more days, whereas those pairs belonged to each treated group including the control group at a low incidence such as one or less in each group.
Fertility indices of females in each treated group including the control group were favorable ranging from 87.5 to 100.0% and there were no statistically significant differences between each treated group and the control group. The numbers of females without gestation were 2 females at 20 ppm and 3 females at 200 ppm.
Gestation indices in each treated group including the control group were 100% except for 95.2% at 200 ppm. In one female which parturition was not confirmed, implantation site was noted in the uterine horns.
Durations of gestation in females in each treated group were similar to those in the control group and almost all females having live pups delivered at Day 22 of gestation. Other remaining females delivered at Day 21 or 23 of gestation.
Numbers of implantation sites in females in each treated group were similar to those in the control group.
The mean numbers of pups delivered in each treated group were similar to those in the control group.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

2 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at this dose level

Remarks on result:

other: equivalent to 0.142 and 0.204 mg/kg bw/day in males and females, respectively

Key result

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

20 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Remarks on result:

other: equivalent to 1.40 and 2.03 mg/kg bw/day in males and females, respectively

Key result

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

>= 200 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: not determinable due to absence of adverse toxic effects

Remarks on result:

other: equivalent to 14.6 and 20.9 mg/kg bw/day in males and females, respectively

Critical effects observed:

yes

Lowest effective dose / conc.:

20 ppm

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Critical effects observed:

yes

Lowest effective dose / conc.:

20 ppm

System:

endocrine system

Organ:

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At observation on Days 15-21 of lactation, opacity of the eyeballs was noted in 2 animals at 200 ppm.
In the treated group, dark-coated skin, concavity of the head, wound of the ear, bending of the tail and loss of fur in the back were noted in one or 2 pups during the lactation period; this was not considered to be treatment-related.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

Not applicable.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

At observation on Day 0 of lactation, there were a few pups found dead in each treated group, whereas there were no statistically significant differences of the incidences between each treated group and the control group. At observation on Days 1-4 of lactation, one pup died at 200 ppm. In addition, there were one to 4 animals with lost of pups in each group including the control group. However, there were no statistically significant differences of the incidences between each treated group and the control group. At observation on Days 5-7 and Days 8-14 of lactation, lost of pups was noted at 2 ppm.
At 20 ppm, one animal with malocclusion and eye discharge died after weaning. At necropsy of the animal, these findings were concluded to be caused by fracture of the nasal bone.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weights of the pups at 2 ppm were similar to those in the control group during the lactation period. At 20 ppm, the body weights of male and female pups were significantly lower than those in the control group in the later half of lactation period. At 200 ppm, the body weights were lower than those in the control group during the lactation period and there were statistically significant differences of the values on Day 7 and thereafter of lactation.

Food consumption and compound intake (if feeding study):

not examined

Description (incidence and severity):

Not applicable.

Food efficiency:

not examined

Description (incidence and severity):

Not applicable.

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

not examined

Description (incidence and severity):

Not applicable.

Ophthalmological findings:

not examined

Description (incidence and severity):

Not applicable.

Haematological findings:

not examined

Description (incidence and severity):

Not applicable.

Clinical biochemistry findings:

not examined

Description (incidence and severity):

Not applicable.

Urinalysis findings:

not examined

Description (incidence and severity):

Not applicable.

Sexual maturation:

effects observed, treatment-related

Description (incidence and severity):

At observation of sexual development, the age at completion of preputial separation in males was delayed at 20 and 200 ppm in relation with the dose levels of test substance and there were statistically significant differences of these values between those treatment groups and the control group. However, the body weights at completion of preputial separation were similar among each group and the control group. On the other hand, the age at completion of preputial separation in males at 2 ppm was similar to that in the control group. In sexual development in females, there were no statistically significant differences of the age and body weights at completion of vaginal opening between each treated group and the control group.

Anogenital distance (AGD):

not examined

Description (incidence and severity):

Not applicable.

Nipple retention in male pups:

not examined

Description (incidence and severity):

Not applicable.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The absolute weights of the liver increased in male and female weanlings in each treated group, similar to their parental animals. The relative weights of these changes were closely related to the dose levels and there were statistically significant differences of the changes between male and female weanlings at 20 and 200 ppm. In addition, the absolute weights of the brain, spleen and thymus of both sexes often showed significant low values at 20 and 200 ppm. However, the relative weights of these organs were similar to or significantly higher than those in the control group, being considered to be the changes caused by low body weights.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At necropsy of pups found dead on Day 0 of lactation, there were no findings except for left umbilical artery in one pup at 2 ppm. At necropsy of pups excluded on Day 4 of lactation, left umbilical artery was noted in 5 to 12 animals in each group including the control group and testicular hemorrhage, hepatodiaphragmatic nodule, white spots on the liver, and thymic remnant in the neck were noted in one to 3 animals in each group including the control group. However, there were no statistically significant differences of the incidences of these findings between each treated group and the control group. At necropsy of weanlings, opacity of the eyeballs and coarsened corneal surface were noted in 22 and 7 pups, respectively, at 200 ppm. However, none of these findings were noted at 2 and 20 ppm. Dilatation of renal pelvis was noted in weanlings in each group including the control group. There was no tendency of increasing incidence of this finding at 2 and 20 ppm. On the other hand, cyst of the ovary, hepatodiaphragmatic nodule, and red sebum were noted occasionally without statistical significance.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

The eyeballs and liver being weighed were examined histopathologically in all weanlings, which revealed keratitis in some weanlings in the groups treated at 20 ppm and above. The incidences of this finding in male weanlings at 20 and in male and female weanlings at 200 ppm were significantly higher than those in the control group. On the other hand, there were no abnormal findings in the eyeballs of any weanlings at 2 ppm. In the liver, focal necrosis of the hepatocyte was observed in one or 2 males and female in each treated group, whereas there were no statistically significant differences of the incidence between each treated group and the control group.

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Behaviour (functional findings):

not examined

Description (incidence and severity):

Not applicable.

Developmental immunotoxicity:

not examined

Description (incidence and severity):

Not applicable.

Sex ratio and the mean viability indices of pups on any lactation days were similar in each treated group to that in the control group.

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

2 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at this dose level

Key result

Dose descriptor:

LOAEL

Remarks:

developmental

Generation:

F1

Effect level:

20 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

sexual maturation

body weight and weight gain

gross pathology

Remarks on result:

other: the delayed preputial separation in males was observed in offspring with a reduced body weight gain during lactation Day 0-7

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

At 200 ppm, intraocular opacity was noted in 2 animals in the same litter, whereas no gross abnormalities were noted in the cornea, considered to be unrelated to the treatment of test substance.
In the treated group, dark-coated skin, concavity of the head, wound of the ear, bending of the tail and loss of fur in the back were noted in one or 2 pups during the lactation period, being not suspected the relation with the treatment of test substance.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

Not applicable.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

At observation on Day 0 of lactation, there were a few pups found dead in each treated group, whereas there were no statistically significant differences of the incidences between each treated group and the control group. At observation on Days 1-4 of lactation, one pup died in the control group. In addition, there were one to 4 animals with loss of pups in each group including the control group and exceptionally all pups were lost in a litter at 2 ppm. However, there were no statistically significant differences of the incidences between each treated group and the control group. At observation on Days 5-7 and Days 8-14 of lactation, loss of pups was noted at 20 ppm.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weights of the pups at 2 ppm were similar to those in the control group during the lactation period. The body weights at 20 ppm were not significantly different from those in the control group. At 200 ppm, the body weights were lower than those in the control group during the lactation period and there were statistically significant differences of the values on Day 7 and thereafter of lactation.

Food consumption and compound intake (if feeding study):

not examined

Description (incidence and severity):

Not applicable.

Food efficiency:

not examined

Description (incidence and severity):

Not applicable.

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

not examined

Description (incidence and severity):

Not applicable.

Ophthalmological findings:

not examined

Description (incidence and severity):

Not applicable.

Haematological findings:

not examined

Description (incidence and severity):

Not applicable.

Clinical biochemistry findings:

not examined

Description (incidence and severity):

Not applicable.

Urinalysis findings:

not examined

Description (incidence and severity):

Not applicable.

Sexual maturation:

no effects observed

Description (incidence and severity):

The absolute values and relative body weight ratio of anogenital distance in male and female pups in each treated group were similar to those in the control group.

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

not examined

Description (incidence and severity):

Not applicable.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The absolute weights of the liver increased in male and female weanlings in each treated group, similar to their parental animals. The relative weights of these changes were closely related to the dose levels and there were statistically significant differences of the changes between male and female weanlings in all treated groups and those in the control group. In addition, the absolute weights of the brain, spleen and thymus of both sexes often showed significant low values at 20 and 200 ppm. However, the relative weights of these organs were similar to or significantly higher than those in the control group, being considered to be the changes caused by low body weights. Furthermore, the relative weight of the brain in male weanlings at 2 ppm was significantly low, whereas there were no significant differences at 20 and 200 ppm, being considered to be an accidental change.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

No abnormalities of pups found dead on Day 0 of lactation were observed at necropsy. At necropsy of weanlings, opacity of the eyeballs and coarsened corneal surface were noted each in 36 pups at 200 ppm. However, none of these findings were noted at 2 and 20 ppm. Dilatation of renal pelvis was noted in weanlings in each group including the control group reaching statistically significance at 200 ppm. There was no tendency of increasing incidence of this finding at 2 and 20 ppm. On the other hand, cyst of the ovary, hepatodiaphragmatic nodule, and red sebum were noted occasionally without statistical significance.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

The eyeballs and liver being weighed were examined histopathologically in all weanlings, which revealed keratitis in some weanlings in the groups treated at 20 ppm and above. The incidences of this finding in male and female weanlings at 200 ppm were significantly higher than those in the control group. On the other hand, there were no abnormal findings in the eyeballs of any weanlings at 2 ppm. In the liver, focal necrosis of the hepatocyte was observed in one or 2 males and female in each treated group, whereas there were no statistically significant differences of the incidence between each treated group and the control group.

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Behaviour (functional findings):

not examined

Description (incidence and severity):

Not applicable.

Developmental immunotoxicity:

not examined

Description (incidence and severity):

Not applicable.

Sex ratio and the mean viability indices of pups on any lactation days were similar in each treated group to that in the control group.

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F2

Effect level:

2 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at this dose level

Key result

Dose descriptor:

LOAEL

Remarks:

developmental

Generation:

F2

Effect level:

20 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

20 ppm (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

The study was performed under GLP conditions and according to OECD 416. The no observed adverse effect level (NOAEL) of the test substance for parental systemic toxicity was concluded to be 2 ppm (P generation: 0.126 mg/kg/day in males and 0.202 mg/kg/day in females, F1 generation: 0.142 mg/kg/day in males and 0.204 mg/kg/day in females), based on the effects found in the thyroid gland and kidneys. No adverse effects on reproductive performance of parental animals were noted even in the highest dose level of 200 ppm in the present study which leads to a NOAEL for fertility of 200 ppm (P generation: 13.1 mg/kg/day in males and 20.4 mg/kg/day in females, F1 generation: 14.6 mg/kg/day in males and 20.9 mg/kg/day in females). Treatment-related effects were only noted in offspring at the same dose levels at which maternal toxicity was observed. Based on reduced body weights, the NOAEL of the test substance in offspring was concluded to be 2 ppm (P generation: 0.126 mg/kg/day in males and 0.202 mg/kg/day in females, F1 generation: 0.142 mg/kg/day in males and 0.204 mg/kg/day in females).

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

13.1 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-X, 8.7, of Regulation (EC) No 1907/2006.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Two studies are available for the test substance that investigate reproductive toxicity, both conducted in rat. The first a pilot study for the two-generation reproductive toxicity study was conducted following the general principles of OECD TG 416 and under GLP. The second a two-generation reproductive toxicity study was conducted in accordance to OECD TG 416 and under GLP.

Pilot study for the two-generation reproductive toxicity study (M-080214-01-1):

The pilot study was conducted in Wistar rats. The test substance was administered to groups of 10 male and 10 female rats at concentrations of 0, 100, 500 or 2500 ppm in their diet (equivalent to 0, 7.3, 38.3 and 195 mg/kg bw/day and 0, 8.6, 48.7 and 305.1 mg/kg bw/day in males and females, respectively, during the pre-mating period). Parental F0 animals were pre-treated over a period of about 4 weeks and allowed to mate over a period of up to three weeks. F1 offspring were nursed up to an age of four weeks.

Mortality, clinical signs, body weights, food intake, mating performances, fertility and duration of pregnancy were examined in F0 rats. Reproduction parameters such as litter size, percentage of males born and pup weight at birth as well as viability, rearing, lactation, body weight gain and clinical signs were studied in F1 offspring. Necropsies were done in all rats. Implantation sites in F0 females were recorded. In F0 parental rats and weanlings selected organs were weighed.

There were no effects on mortality and behaviour of the F0 parental animals at levels of up to 2500 ppm. However, there was one 2500 ppm male exhibiting eye opacity. Body weights of treated F0 rats were reduced dose-independently by about 3-11% (p≤0.05) from 100 ppm onwards. In F0 females, increased food intake was noted from 500 ppm onwards resulting in a higher substance intake than expected from the theoretical dose factor.

There were partly significantly higher relative weights of the liver and kidneys in F0 males from 100 ppm onwards, however, without a dose dependency. No treatment related gross lesions were noted in F0 rats up to 2500 ppm.

The reproduction parameters insemination index, mating performance, fertility index, gestation index, duration of pregnancy, life birth index, birth weights and percentages of males born, prenatal loss and litter size were not affected at levels of up to 2500 ppm. The number of implantation sites and pups born were not remarkably altered up to 500 ppm and slightly reduced at 2500 ppm.

The pup weight development was retarded mostly statistically significantly and dose dependent in all treatment groups from Day 7 post-partum onwards. This resulted in partly statistically significantly reduced weights of the brain, spleen and thymus. The viability, rearing and lactation indices were not affected up to 2500 ppm. Clinical observation of F1 pups revealed no remarkable changes up to 500 ppm and an increased number of thin pups at 2500 ppm. No test substance-related gross pathological findings were observed in F1 offspring up to 2500 ppm. The skeletal development of the pups or weanlings was unaffected at dose levels of up to 2500 ppm.

Thus, because of the effects on body weights of parental rats and their pups at all dose levels a no-observed-adverse effect level (NOAEL) could not be established in this study.

Two-generation reproductive toxicity study (M-399182-01-2):

The test substance was mixed in the basal feed at 0 (basal feed), 2, 20, and 200 ppm and given to 24 male and 24 female SPF Wistar Hannover rats/group for two successive generations, to evaluate the potential effects on reproductive performance and on the growth and development of their offspring.

Systemic toxicities of the test substance on parental animals were observed at a dose level of 20 ppm and above. In these dose groups, opacity of the eyeballs and/or coarsened corneal surface were found in male and female parental animals, which caused statistically significant increases in the incidence of opacity of the eyeballs in P males and that of both findings in F1 males and in P and F1 females. After histopathological examination these effects were diagnosed as keratitis. Similar effects were observed in the combined repeated dose toxicity/carcinogenicity study (M-270092-01-1). These findings are characteristics of a compound such as the test substance that inhibits 4-ydroxyphenylpyruvic acid dioxygenase (4-HPPDase), an enzyme of the tyrosine catabolic pathway. These lesions are related to an increase in plasma tyrosine level caused by a blockade of the 4-HPPDase enzyme in the rat. However, the rat is a species particularly sensitive to inhibition of the 4-HPPDase enzyme and is atypical in its susceptibility to develop tyrosine-related eye lesions. Therefore, although these lesions (corneal lesions and eye keratitis) were treatment-related, they were considered not to be toxicologically relevant to man. This is discussed further within the repeated dose toxicity endpoint summary.

Body weights, body weight gains and food consumption of parental animals in these treated groups were also affected by the test substance treatment, in which significantly lower-than-control values were found in body weights of the 200 ppm group and body weight gains and food consumptions of the 20 and 200 ppm groups.

The following observations were made on organ weights and histopathological parameters:

A significant increase in relative thyroid weights of P males in the 20 and 200 ppm groups was accompanied by such histopathological alterations as increased large-sized follicles, colloidal alteration and hypertrophy of the follicular cell and the incidences of these findings in both treated groups (20 and 200 ppm treated groups) were statistically significantly higher than those in the control group. As the incidences of colloidal alteration in these groups were also higher than that in the control group in F1 generation, the test substance is concluded to have an effect on the thyroid at the dose levels higher than 20 ppm, although an increase of the thyroid weight was unclear in the F1 generation. The weight of the thyroid in males of the 2 ppm group was comparable to that in the control group and there was no statistically significant difference in the incidence of histopathological findings of the thyroid between the control and 2 ppm groups. Thyroid effects related to test substance administration are discussed further within the repeated dose toxicity endpoint summary.

A significant increase in relative kidney weights of parental males at a dose level of 20 ppm and above are thought to be treatment-related because of the clear dose- response relationship in both generations. Histopathological observations also revealed chronic nephropathy in two F1 males out of 12 examined in the 200 ppm group. This change is suggested to be related to the test substance treatment because chronic nephropathy rarely occurred in young rats and an increase in kidney weights was accompanied by a significantly increased incidence of chronic nephropathy in the treated groups at 50 ppm and above in the previous “Chronic toxicity and carcinogenicity study of the test substance in the Wistar rat by dietary administration” (M-270092-01-1). In the 2 ppm group, however, neither kidney weights nor histopathological changes were observed.

Absolute and/or relative adrenal weights increased significantly in F1 male and female parental animals of the treated groups at 20 ppm and above when compared to those in the control group. However, this does not suggest toxicologically significant effects of the test substance since histopathological observations of 12 selected animals in the 200 ppm group revealed no abnormalities in the adrenals. There were no statistically significant differences of these organ weights between the 2 ppm group and the control group.

Relative liver weights of both males and females in the treated groups at 20 ppm and above were significantly and dose-dependently higher than those in the control group in both generations. The histopathological examinations of all the parental animals showed an increased incidence of centrilobular hypertrophy of the hepatocyte in males of the treated groups at 20 ppm and above. These results suggest that the increase in liver weight is a physiological response to metabolizing a xenobiotic. Furthermore, an increase in liver weights in the 200 ppm group, in which a degree of weight change was most obvious, was relatively slight (less than 130% to the control group), leading to the conclusion that a series of alterations observed in the liver are not adverse but merely adaptive.

Relative brain weights of the females in the 200 ppm group, as well as absolute brain weights of F1 male and female parental animals in the 20 and 200 ppm groups were significantly lower than those in the control group, respectively. Similar alterations were also found in F1 weanlings of the 200 ppm group, in which absolute brain weights decreased significantly while relative weights increased significantly. These results suggest that decreases in brain weights are due to suppressed body weights in these F1 parental animals. In the present study, suppression of body weight gains in females selected for F1 female parents was limited during a first half of the growth period and the lactation period and final body weights at necropsy in the treated groups were comparable to those in the control group. These facts lead to the speculative interpretation that relative brain weight of F1 parental females seem to be reduced at necropsy due to slight suppression of brain growth during early life accompanied by normal body growth in the later life. Thus, the decrease in brain weight is considered to be a non-specific alteration which was caused mainly by the treatment-related suppression of body weight gains.

In the P-males, the relative weights of the testes in all treated groups increased significantly, being suspected to be related to the treatment of test substance. In addition, the absolute weights of the epididymides and seminal vesicles increased at 20 and 200 ppm. In the P1-males the relative weights of the testes at 20 and 200 ppm increased significantly, compared with control. In addition, the absolute weights of the epididymides and seminal vesicles at 20 and 200 ppm were slightly higher than those in the control group. The relative weights of the seminal vesicles at 200 ppm reached statistically significance. However, these were thought to be toxicologically insignificant since no abnormalities were seen in such parameters as the number of sperm, sperm motility and reproductive performance, and histopathological observations failed to reveal findings that may be related to the organ weight increases. Furthermore, the relative weight of testes and epididymides generally increases in rats with decreased body weight (Derelanko, M.J. Toxicologist’s pocket handbook. 2003 ISBN 0-8493-0009-6). In conclusion, these effects were not considered to be relevant to the fertility of the male rats.

Swelling of the mammary gland in parental females in the 20 and 200 ppm groups after weaning are suggested to be related to the test substance treatment because the findings are concordantly observed in both P and F1 generations, although their incidences (1 to 3 animal/group) were not statistically significantly different from that in the control group. Histopathology of the mammary gland of these females with gross findings revealed incomplete recovery from lactation phase. The alterations found here seem to be toxicologically insignificant and judged as not adverse.

In reproductive performance, there were no changes related to the test substance treatment in the following reproductive parameters: incidence of females with normal oestrous cycle, mating index, fertility index, duration of gestation, and the number of implantation sites in parental animals. In sperm examination for P and F1 parental male animals, sperm head count in the testis, sperm counts in the cauda epididymis and sperm motility in each group were not significantly different from those in the control group. The histopathological examination of the reproductive organs and pituitary showed no abnormal findings in these animals.

Effects of the test substance treatment on pups were noted as suppression of body weight gains in males and females at a dose level of 20 ppm and above. Opacity of the eyeballs was also found grossly in weanlings of the 200 ppm group with a low incidence and pathological observation revealed keratitis in some animals of the 20 and 200 ppm groups. However, these findings are considered not to be relevant to humans. No abnormal findings were detected in pups of the 2 ppm group in terms of body weights and eyeballs in both generations. At necropsy, the incidence of dilatation of renal pelvis increased significantly in F2 weanlings in the 200 ppm group. In addition, relative liver weights increased significantly in weanlings of all treated groups. However, these weight changes are considered not to be toxicologically significant because they were accompanied by no histopathological alterations. Significantly lowered absolute weights of the brain, spleen and thymus at 20 ppm and above are also thought to be sporadic fluctuations related to a suppression of body weights.

Based on the effects found in the thyroid gland and kidneys, the no observed adverse effect level (NOAEL) of the test substance in parental rats regarding the parental systemic toxicity was concluded to be 2 ppm (P generation: 0.126 mg/kg/day in males and 0.202 mg/kg/day in females, F1 generation: 0.142 mg/kg/day in males and 0.204 mg/kg/day in females). Based on reduced body weight gain in male and female F1 pups at a dose level of 20 ppm and above, the NOAEL for offspring toxicity is the same as for parental toxicity.

No adverse effects on reproductive performance of parental animals were noted even in the highest dose level of 200 ppm in the present study which leads to a NOAEL for fertility of 200 ppm (P generation: 13.1 mg/kg/day in males and 20.4 mg/kg/day in females, F1 generation: 14.6 mg/kg/day in males and 20.9 mg/kg/day in females).

Effects on developmental toxicity

Description of key information

Developmental toxicity (rat, OECD 414):

NOAEL (maternal systemic): 1 mg/kg bw/day

NOAEL (developmental): 1 mg/kg bw/day

NOAEL (teratogenicity): >1000 mg/kg bw/day

Developmental toxicity (rabbit, OECD 414):

NOAEL (maternal systemic): 10 mg/kg bw/day

NOAEL (developmental): 0.1 mg/kg bw/day

NOAEL (teratogenicity): > 1000 mg/kg bw/day

Two-Generation Toxicity Study (OECD 416), rats:

NOAEL systemic = 2 ppm corresponding to:

NOAEL systemic (males, P1) = 0.126 mg/kg bw/day

NOAEL systemic (females, P1) = 0.202 mg/kg bw/day

NOAEL systemic (males, F1) = 0.142 mg/kg bw/day

NOAEL systemic (females, F1) = 0.204 mg/kg bw/day

NOAEL (developmental) = 2 ppm corresponding to a dietary intake of parents (P and F1 parental animals):

NOAEL developmental (males, P1 generation) = 0.126 mg/kg bw/day

NOAEL developmental (females, P1 generation) = 0.202 mg/kg bw/day

NOAEL developmental (males, F1 generation) = 0.142 mg/kg bw/day

NOAEL developmental (females, F1 generation) = 0.204 mg/kg bw/day

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Feb - 20 Jul 2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

Results tables are missing in the translated version of the study report.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 22 Jan 2001

Deviations:

yes

Remarks:

results tables are missing in the translated study report

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 25 Jun 2018

Deviations:

yes

Remarks:

Please refer to "Principles of method if other than guideline".

Principles of method if other than guideline:

Deviations to OECD guideline 414 (2018): Diet not analysed (phytoestrogens); no investigations of thyroid weight and histopathology; reproductive tract of fetuses or cryptorchidism not examined; no comparison between external vs. internal (gonadal) sex morphology; historical control data from the testing facility not included; high dose level (1000 mg/kg bw/day) induced mortality (6/25 animals), therefore exceeding the maximum tolerated dose.

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

other: Japanese White rabbits (Kbl:JW)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: KITAYAMA LABES Co., Ltd. (Nagano, Japan)
- Age at study initiation: 18 weeks
- Weight at study initiation: 3.10 - 3.74 kg on receipt
- Fasting period before study: No
- Housing: Individually housed in aluminium cages with wire mesh floors (W350 x D480 x H330 mm)
- Diet: Certified solid feed LRC4 (Oriental Yeast Co., Ltd., Tokyo, Japan), ad libitum
- Water: Purified well water, ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

other: aqueous solution of 0.5% methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosing solution was prepared for each dose level by suspending a specified amount of the test substance in an aqueous solution of 0.5% methylcellulose.

VEHICLE
- Concentration in vehicle: 0.002 to 20%
- Amount of vehicle (if gavage): 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

All samples were analysed by using a high performance liquid chromatograph (HPLC) LC-2000 Plus (JASCO Corp., Tokyo, Japan) with L-column ODS (Chemicals Evaluation and Research Institute, Japan, Tokyo, Japan).

Prior to the study, a stability study was performed for the low dose group (0.02 mg/mL). The stability of test substance at concentrations of 0.1 mg/mL and 200 mg/mL had already been confirmed in a previous study (SR04179, 2005), in which the test substance was stable for 15 days under 6-hour room temperature and subsequent refrigerated condition. The stability study revealed that the test substance was stable for 14 days after preparation when stored under refrigerated conditions (100% recovery).

Dosing solutions were prepared 4 times with intervals of 7 days during the study period. Analysis of the dose preparations confirmed that they were homogenous and the test substance concentrations were within appropriate ranges.

In the homogeneity study, the coefficient of variation (CV) of the dosing solutions in the low and high dose groups was 0.0% and 1.6%, respectively, demonstrating that the test substance was uniformly distributed in the dosing solutions.

In the concentration analysis, the test substance was detected in the samples from each dosing solution at a range of 97% to 105% of the nominal concentrations, indicating that the preparation of dosing solutions had been properly performed.

Details on mating procedure:

- Impregnation procedure: Artificial insemination

Semen was taken from 2-4 males per day and those with good quality were pooled and diluted with physiological saline at a dilution of 1:10. Each female was injected 0.5 mL of the diluted semen into the vagina. After the injection, the females were given 25 U of hCG (human Chorionic Gonadotropin) through the auricular vein. These mating procedures were repeated on 20 females per day for 5 consecutive days.

- Verification of same strain and source of both sexes: Yes

Duration of treatment / exposure:

22 days (gestation Day 6 to 27)

Frequency of treatment:

Daily, 7 days a week

Duration of test:

Day 6 to 27 of gestation

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In a dose range-finding study (IET 04-0029, 2006), the test substance was administered orally to artificially inseminated Japanese White rabbits (Kbl:JW), eight females per group, once daily from Days 6 to 27 of gestation at doses of 0, 1, 10, 100, and 1000 mg/kg bw/day to examine the potential effects on maternal animals and their fetuses. The study revealed no adverse effects of the test substance on maternal body weights and food consumptions, as well as the number of surviving fetuses and fetal body weights in any of the treated groups. In the skeletal examination, incidences of fetuses with supernumerary ribs and 27 presacral vertebrae in all dose groups were significantly higher than those in the control group. The incidences of fetuses with supernumerary ribs and 27 presacral vertebrae were 52.8% and 39.6%, respectively, in the 1 mg/kg bw/day group, whereas those in the 10 mg/kg bw/day and higher dose groups ranged from 90.2% to 91.0% and from 80.4% to 88.9%, respectively (29.8% and 14.9%, respectively in the control group). None of the malformations observed were thought to be treatment-related in all dose groups. Based on the results described above, a dose level of 0.1 mg/kg bw/day was selected as the low dose level, with the expectation that the incidences of supernumerary ribs and 27 presacral vertebrae in fetuses will be similar to those in the control group, and those of 10 mg/kg bw/day and 1000 mg/kg bw/day were selected as the middle and high dose levels, respectively.
- Rationale for animal assignment: Artificially inseminated females were weighed on Day 0 of gestation (the day of artificial insemination) and distributed to 4 groups in such a way to equalise group mean body weights as closely as possible.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily during the dosing period

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were performed on the days of body weight measurement (Days 0, 6, 9, 12, 15, 18, 21, 24, 27 and 28 of gestation)

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 6, 9, 12, 15, 18, 21, 24, 27 and 28 of gestation. Adjusted body weights were calculated by subtracting the gravid uterine weight from the body weight on Day 28 of gestation.

FOOD CONSUMPTION: Yes
- The amount of food supplied and/or unconsumed was determined for each animal on Days 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 28 of gestation. Daily food consumption (g/rabbit/day) was calculated for each female by dividing the amount of total food consumption by the number of feeding days.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation Day 28 followed by a gross pathological examination. Organs and tissues were not preserved.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

When no uterine implants were apparent upon gross examination, the uterus was stained with 10% ammonium sulfide solution to detect very early resorptions.

Blood sampling:

- Plasma: No
- Serum: No

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes, half of the animals underwent fresh examination of eyes and brains, the heads of the other half were preserved for observations of eyes, brain, nasal passage and tongue.
-Determination of sex by observation of the internal reproductive organs

After soft tissue examination, these soft tissues (thoracic and abdominal) were removed and preserved in Bouin's solution with the placentas and the examined eyes (half of litter). The heads of the other half of the fetuses was decapitated along with the rima oris and fixed in Bouin's solution. The remaining skeletons were fixed in 70% isopropyl alcohol solution, stained with alizarin red S, and cleared in 70% glycerin for examination of skeletal abnormalities.

Statistics:

See field "Any other information on materials and methods incl. tables".

Indices:

Percent incidences of preimplantation losses were calculated from the following formula:

Percent incidence of preimplantation losses (%) =
((Number of corpora lutea – number of implants) / number of corpora lutea) x 100

The sex ratio was calculated for each group from the following formula:
Sex ratio = total number of male fetuses / total number of surviving fetuses

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No clinical abnormalities were noted in any maternal rabbits in each treated group during the pre-dosing period (Days 0 to 5 of gestation).

During the dosing period (Days 6 to 27 of gestation), five animals at 1000 mg/kg bw/day (one of them was non-pregnant) died between Days 20 to 27 of gestation, and the incidence was significantly higher than that in the control group. These animals showed such clinical signs as soiled fur and red discharge on the tray and one of them aborted before death. At 10 mg/kg bw/day, posterior paralysis was noted in one animal on Day 12 of gestation. This animal was euthanatized because the lesion was determined to non-recoverable. In the control group, one animal aborted on Day 26 of gestation. In addition, loss of fur or crust formation was noted in one or two animals in the control and at 0.1 mg/kg bw/day.

Observations after the termination of administration (Day 28 of gestation) revealed death of an animal (non-pregnant) at 1000 mg/kg bw/day accompanied by the presence of soiled fur and emaciation. Red discharge on the tray was noted in another animal in the same group. There were no abnormal findings in any animals in the control, 0.1 and 10 mg/kg bw/day groups.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

Not applicable.

Mortality:

mortality observed, treatment-related

Description (incidence):

Five animals at 1000 mg/kg bw/day died between gestation Days 20 and 27. One female at 1000 mg/kg bw/day died on gestation Day 28.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were no statistically significant differences in the mean body weights and the adjusted body weight at cesarean section of maternal animals between the control group and the 0.1, 10 and 1000 mg/kg bw/day groups.

The mean body weight gains in the 0.1 and 10 mg/kg bw/day groups were comparable to those in the control group during the study period. At 1000 mg/kg bw/day, the mean body weight gains were significantly lower than those in the control group on gestation Day 15 and thereafter.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The mean food consumption in the 0.1 and 10 mg/kg bw/day groups was comparable to that in the control group during the study period. At 1000 mg/kg bw/day, the mean food consumption of females was suppressed slightly after the initiation of treatment and the values during gestation Days 6 to 9 and 15 to 18 were significantly lower than those in the control group.

Food efficiency:

not examined

Description (incidence and severity):

Not applicable.

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

not examined

Description (incidence and severity):

Not applicable.

Ophthalmological findings:

not examined

Description (incidence and severity):

Not applicable.

Haematological findings:

not examined

Description (incidence and severity):

Not applicable.

Clinical biochemistry findings:

not examined

Description (incidence and severity):

Not applicable.

Endocrine findings:

not examined

Description (incidence and severity):

Not applicable.

Urinalysis findings:

not examined

Description (incidence and severity):

Not applicable.

Behaviour (functional findings):

not examined

Description (incidence and severity):

Not applicable.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The mean gravid uterine weights in the 10 and 1000 mg/kg bw/day groups were comparable to those in the control group. Although the mean gravid uterine weight in the 0.1 mg/kg bw/day group was significantly higher than that in the control group, this is likely to be due to a slightly increased fetal number in this group and is considered to be unrelated to the test substance treatment.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Animals subjected to necropsy during the study included six females that died at 1000 mg/kg bw/day, one female at 10 mg/kg bw/day that was euthanatized due to posterior paralysis and one female in the control group that aborted. Among them, two females out of 6 at 1000 mg/kg bw/day were non-pregnant and were excluded from the evaluation. The necropsy revealed 1 - 3 cases of soiled fur in the perinasal/perianal regions, hair bolus in the stomach, distention of the stomach, watery contents in the caecum, spots or red in color of the lungs and haemorrhage in the uterine horn in four dead females at 1000 mg/kg bw/day. In addition, one female euthanatized at 10 mg/kg bw/day had dislocation in the lumbar vertebra and one aborted female in the control group exhibited watery contents in the caecum and haemorrhage in the uterine horn.

No gross abnormal findings were observed in any animals euthanatized on Day 28 of gestation.

Neuropathological findings:

not examined

Description (incidence and severity):

Not applicable.

Histopathological findings: non-neoplastic:

not examined

Description (incidence and severity):

Not applicable.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

Not applicable.

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

Of the six animals that died at 1000 mg/kg bw/day, one aborted before death.

In the control group, one animal aborted on Day 26 of gestation.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no statistically significant differences in the mean numbers of corpora lutea, implants and the mean percent incidences of preimplantation losses between control and treated groups.

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

Two females in the control group, three females at 0.1 mg/kg bw/day, one female at 10 mg/kg bw/day and one female at 1000 mg/kg bw/day had no grossly observable conceptus at cesarean sectioning. However, the staining of these uteri with 10% ammonium sulfide solution revealed that all these females except for one at 10 mg/kg bw/day group were pregnant because of the presence of staining-positive implantation vestiges.

All the data obtained from females with no grossly observable conceptus were excluded from the calculation of group mean values. Consequently, the numbers of maternal animals with live fetuses at cesarean sectioning were 22, 22, 23 and 18, respectively, in the control, 0.1, 10 and 1000 mg/kg bw/day groups.

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Not applicable.

Changes in number of pregnant:

no effects observed

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Details on maternal toxic effects:

Maternal toxicity was noted in the 1000 mg/kg bw/day group, in which six animals (including two non-pregnant animals) died between gestation Days 20 to 28 after showing a decrease in food consumption and/or a stop of feeding with decreased body weights. Five of them (including two non-pregnant animals) also exhibited such clinical findings as soiled fur and/or red discharge on the tray and one aborted on the day of death. The mean body weight gains on gestation Day 15 and thereafter, as well as the mean food consumption during gestation Days 6 to 9 and 15 to 18 in this group were significantly lower than those in the control group.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: No adverse effects seen at this dose level.

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

mortality

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean weights of female fetuses were comparable to those in the control group. The mean weights of male fetuses at 10 and 1000 mg/kg bw/day were significantly lower than that in the control group.

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

The mean numbers of live fetuses at 10 and 1000 mg/kg bw/day were comparable to that in the control group. The mean number of live fetuses at 0.1 mg/kg bw/day was significantly higher than that in the control group. However, this was not thought to be toxicologically significant.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Description (incidence and severity):

Not applicable.

Changes in postnatal survival:

not examined

Description (incidence and severity):

Not applicable.

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal observations revealed that one and two fetuses in the control and 0.1 mg/kg bw/day groups, respectively, had fused sternebrae, one fetus each exhibited absent rib (12th left), supernumerary lumber vertebra (right), and absence of lumbar arch (right) and centrum (4th) in the control group, and two and one fetus in the control and 1000 mg/kg bw/day groups, respectively, had misaligned caudal centrum. However, these abnormalities are considered to be unrelated to the test substance treatment since their incidences were low.

For skeletal variations, the incidences of supernumerary rib and 27 presacral vertebrae in the 10 and 1000 mg/kg bw/day groups were significantly higher than those in the control group. These findings are considered to be related to the treatment of test substance since similar changes were also found in the preliminary range-finding study. Significantly lowered incidence of cervical rib was seen in the 10 mg/kg bw/day group, but was not considered to be toxicologically significant. In addition, some fetuses in all groups including the control exhibited incomplete ossification of interparietal bones, misaligned sternebra, extra sternebral ossification site (between the 5th and 6th sternebrae), incomplete ossification of cervical arch (1st, right), incomplete ossification of cervical centrum (3rd or 4th), lumbosacral transitional vertebra and bipartite ossification of caudal centrum. However, there were no statistically significant differences in the incidences of these findings between the control and any of the treated groups.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

In the visceral observations, one fetus each in the control group exhibited cutis aplasia on the head, narrowed pulmonary artery accompanied by membranous ventricular septum defect, narrowed right subclavian artery accompanied by membranous ventricular septum defect and hydronephrosis, narrowed left common carotid artery and lobation anomaly of the liver. Abnormalities found in the 10 mg/kg bw/day group were one case each of malpositioned kidney, thickened peritoneum around the kidney and fusion of the intestine and bladder. In the 1000 mg/kg bw/day group, one fetus showed misshapen cerebellum and another had a small spleen. However, these abnormalities are considered to be unrelated to the test substance treatment since their incidences were low.

Malpositioned testis was observed in 0, 1, 5 and 6 fetuses in the control, 0.1, 10 and 1000 mg/kg bw/day groups, respectively, and the incidence of this finding in the 10 mg/kg bw/day group was significantly higher than that in the control group. However, the relationship between the increased incidence of malpositioned testis and the test substance treatment is unclear because this abnormality was not found in the rangefinding study in rabbits, nor in the previous teratogenicity study (M-400385-01-2, 2006) and two-generation reproductive toxicity study (M-399182-01-2, 2007) in rats. Additionally, malpositioned testis have been seen in control groups at low incidence in previously conducted teratogenicity studies using the same strain of rabbit, and the frequency seen in the present study (3.0 and 3.7%, in the 10 and 1000 mg/kg bw/day, respectively) falls within the range (0 - 4.4%) published in literature for this strain of rabbit (Nakatsuka et al., Cong. Anom., 37 (1): 47-138, 1997).

Visceral variations observed in this study included malpositioned left common carotid branch, malpositioned right subclavian branch, malpositioned internal thoracic branch and thymic remnant in the neck. There were no statistically significant differences between the control and any of the treated groups in the incidences of these findings.

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Details on embryotoxic / teratogenic effects:

Reduced fetal weights and increased skeletal variations at 10 and 1000 mg/kg bw/day. None of the findings in the study were considered severe enough to be indicative of a teratogenic effect.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects seen at this dose level.

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

other: Increased incidence of skeletal variations

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: supernumerary rib

skeletal: vertebra

Description (incidence and severity):

For skeletal variations, the incidences of supernumerary rib and 27 presacral vertebrae in the 10 and 1000 mg/kg bw/day groups were significantly higher than those in the control group.

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

References

Toshio Nakatsuka et al., Japan Pharmaceutical Manufacturers Association (JPMA) survey on background control data of developmental and reproductive toxicity studies in rats, rabbits and mice. Cong. Anom., 37 (1): 47-138, 1997.

Conclusions:

The study was performed under GLP conditions and according to OECD 414. The test substance caused reduced body weight gain and food consumption, adverse clinical findings and mortality in maternal animals at 1000 mg/kg bw/day, therefore the maternal NOAEL was 10 mg/kg bw/day. Reduced fetal weights in males and increased skeletal variations in males and females were seen at 10 and 1000 mg/kg bw/day, therefore the fetal NOAEL was 0.1 mg/kg bw/day. It was concluded that none of the findings were indicative of a teratogenic effect.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Jun - 25 Sep 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

Results tables are missing in the translated version of the study report.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 22 Jan 2001

Deviations:

yes

Remarks:

results tables are missing in the translated study report

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 25 Jun 2018

Deviations:

yes

Remarks:

Please refer to "Principles of method if other than guideline".

Principles of method if other than guideline:

Deviations to OECD guideline 414 (2018): Diet not analysed (phytoestrogens); no investigations on the thyroid (weight, histopathology, no T4, T3/TSH levels measured); anogenital distance of pups not measured; reproductive tract of fetuses or cryptorchidism not examined; no comparison between external vs. internal (gonadal) sex morphology; historical control data from the testing facility not included.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: BrlHan:WIST@Jcl (GALAS)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Clea Japan Inc. (Fuji Rearing Farm)
- Age at study initiation: Approximately 14 weeks
- Weight at study initiation: At the time of receipt, body weights ranged from 286 to 346g (males) and 165 to 226g (females)
- Housing: One rat per cage, metallic bracket-type cage, with wire mesh floors
- Diet: Gamma-ray irradiated pellet diet (CRF-1, Oriental Yeast Co., Ltd.), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 to 24
- Humidity (%): 47 to 70
- Air changes (per hr): 10 to 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

other: 0.5% methylcellulose aqueous solution

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared twice during the study period. The test substance was weighed and suspended in a 0.5% methylcellulose aqueous solution.

VEHICLE
- Concentration in vehicle: 0.01, 0.3 and 10%, respectively
- Amount of vehicle: 10 mL/kg bw
- Lot/batch no.: Methylcellulose (Lot No. 008614), purified water (Lot No. 503073)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of the dose formulations was conducted using HPLC-UV. Analysis of homogeneity and stability of the dose formulations was conducted as part of a range-finding study (study no. SR04179, 2005). Analysis confirmed the homogeneity and storage stability for 6 hours at room temperature and for 15 days under refrigerated conditions for preparations at 0.1 mg/mL and 200 mg/mL. Based on this analysis, dose formulations were prepared on two occasions as part of the main study. Analysis was conducted on both dose preparations, with all samples in the range of 94.8 to 102.0% of the target concentrations, with a maximum CV of 4.6%. This met the acceptability criteria of 90 – 110% target concentration and CV <5%.

Details on mating procedure:

- Impregnation procedure: Cohoused
- M/F ratio per cage: 1 to 1
- Length of cohabitation: Overnight, mating procedures were repeatedly conducted for 7 days until proof of successful copulation.
- Verification of same strain and source of both sexes: Yes
- Proof of pregnancy: Vaginal plug and / or sperm in vaginal smear, referred to as Day 0 of gestation.

Duration of treatment / exposure:

Day 6 to 19 of gestation.

Frequency of treatment:

Once daily, 7 days/week

Duration of test:

20 days

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on the results of a range finding study (study no. SR04179, 2005), in which animals were orally exposed to 1, 10, 100 and 1000 mg/kg bw/day from gestation Days 6 to 19. Soiling of perigenital fur, the suppression of body weight gain and lower food consumption were observed in the 1000 mg/kg bw/day group. In the 100 mg/kg bw/day group, lowered food consumption was noted after initiation of dosing (although this was not statistically significant). Fetal skeletal variations and the delayed ossification were observed in the 100 and 1000 mg/kg bw/day groups. In the 1000 mg/kg bw/day group, male and female fetal body weights were significantly decreased. On the basis of the range finder results, 1000 mg/kg bw/day was set as the high dose which was expected to induce maternal toxicity. The low dose was set at 1 mg/kg bw/day, which was predicted to cause no toxic effect on maternal animals and fetuses. The mid dose was set at 30 mg/kg bw/day which was the geometric mean.
- Rationale for animal assignment: On each day of successful copulation, mated females were assigned to the 4 groups, (control, low mid and high), so that means and standard deviations of body weights would be equal between the groups.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations were limited to death and general appearance

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Day 0 and 3 of gestation, then daily from Day 6 to 20. In addition, adjusted body weights were calculated by subtracting gravid uterine weight from the body weights on Day 20 of gestation

FOOD CONSUMPTION: Yes
- Food consumption was measured on Days 0, 3, 6, 9, 12, 15, 18, and 20 of gestation. The daily food consumption (g/day) of each animal was calculated by dividing the total food consumption by the number of feeding days

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation Day 20
- Organs examined: All organs and tissues were macroscopically examined. The following organs and tissues were fixed and preserved in 10% neutral-buffered formalin. The brain (cerebrum and cerebellum), hypophysis, thymus, thyroid (bilateral), parathyroid gland (bilateral), adrenal gland (bilateral), spleen, heart, liver, lung (including bronchi), kidneys (bilateral), ovaries (bilateral), uterus (bilateral uterine horn and uterine cervix), vagina, mammary gland (right abdomen) and macroscopic abnormal lesion (including the boundary with the normal tissues)

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Number of live and dead fetuses

Uteri that appeared non-gravid were stained with 10% ammonium sulfide aqueous solution to confirm the non-pregnant status.

Blood sampling:

- Plasma: No
- Serum: No

Fetal examinations:

- External examinations: Yes, all per litter
- Soft tissue examinations: Yes, half per litter
- Skeletal examinations: Yes, half per litter
- Head examinations: Yes
- Anogenital distance of all live rodent pups: No

One half of the live fetuses in each litter were fixed in Bouin's solution for visceral examination. The remaining half of the fetuses were eviscerated and fixed in 70% ethanol for skeletal examination. They were further treated with alizarin red S and alcian blue for bon-cartilage double staining, immersed in 80% glycerin, and transparent skeletal specimen were prepared.

Statistics:

See field "Any other information on materials and methods incl. tables".

Indices:

Uterine indices:
- Implantation index (%) = (Number of implantations / Number of corpora lutea) × 100
- Viability index of fetuses (%) = (Number of live fetuses / Number of implantations) × 100
- Incidence of dead or resorbed embryos and fetuses (%) = (Number of dead or resorbed embryos and fetuses / Number of implantations) × 100
- Sex ratio = Number of live male fetuses / Number of live fetuses

External indices:
- Incidence of fetal external anomalies (%) = (Number of fetuses with external anomalies / Number of fetuses examined) × 100
- Incidence of litters with fetal external anomalies = Number of litters containing fetuses with external anomalies / Number of litters examined

Soft tissue indices:
- Incidence of fetal visceral variations (%) = (Number of fetuses with visceral variations / Number of fetuses examined) × 100
- Incidence of fetal visceral anomalies (%) = (Number of fetuses with visceral anomalies / Number of fetuses examined) × 100
- Incidence of litters with fetal visceral variations = Number of dams having fetuses with visceral variations / Number of litters examined
- Incidence of litters with fetal visceral anomalies = Number of dams having fetuses with visceral anomalies / Number of litters examined

Skeletal indices:
- Incidence of fetal skeletal variations (%) = (Number of fetuses with skeletal variations / Number of fetuses examined) × 100
- Incidence of fetal skeletal anomalies (%) = (Number of fetuses with skeletal anomalies / Number of fetuses examined) × 100
- Incidence of litters with fetal skeletal variations = Number of dams having fetuses with skeletal variations / Number of litters examined
- Incidence of litters with fetal skeletal anomalies = Number of dams having fetuses with skeletal anomalies / Number of litters examined

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Maternal animals did not show any changes in general appearance in the 1 and 30 mg/kg bw/day groups, as well as the control group during the dosing period.

In the 1000 mg/kg bw/day group, soiling of perigenital fur was seen in 21 females (including one non-pregnant rat) out of 24 from Day 7 through Day 20 of gestation. A total of nine females in this group (including one non-pregnant rat) also showed alopecia (breast, abdomen, lumbar part, fore or hind legs) from Day 10 through to Day 20 of gestation.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

Not applicable.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body Weight:
No significant difference was observed in mean body weights and adjusted body weights of maternal animals in the treated groups when they were compared to those in the control group.

Body Weight Gain:
1 mg/kg bw/day:
No significant difference was observed in mean maternal body weight gains of the 1 mg/kg bw/day group when they were compared to that of the control group.

30 mg/kg bw/day:
Significantly lowered values were seen on gestation Days 0 - 10 and 0 - 12.

1000 mg/kg bw/day:
Significantly lowered values were found during gestation Days 0 - 10 and through gestation Days 0 - 20. In addition, maternal body weight gains in this group were significantly suppressed during the dosing period (Days 6 - 20 of gestation).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

No significant difference was seen in mean maternal food consumption of the 1 and 30 mg/kg bw/day groups when compared to that of the control group.

1000 mg/kg bw/day:
Significantly lowered values were noted during gestation Days 6-9.

Food efficiency:

not examined

Description (incidence and severity):

Not applicable.

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

not examined

Description (incidence and severity):

Not applicable.

Ophthalmological findings:

not examined

Description (incidence and severity):

Not applicable.

Haematological findings:

not examined

Description (incidence and severity):

Not applicable.

Clinical biochemistry findings:

not examined

Description (incidence and severity):

Not applicable.

Endocrine findings:

not examined

Description (incidence and severity):

Not applicable.

Urinalysis findings:

not examined

Description (incidence and severity):

Not applicable.

Behaviour (functional findings):

not examined

Description (incidence and severity):

Not applicable.

Immunological findings:

not examined

Description (incidence and severity):

Not applicable.

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Necropsy of maternal animals on gestation Day 20 revealed dilatation of renal pelvis in 1, 1, 1, and 2 females of the control, 1, 30 and 1000 mg/kg bw/day groups, respectively. Other findings observed were one case of white granules in the renal pelvis in the 1 mg/kg bw/day group and the followings in the 1000 mg/kg bw/day group: two cases of white granules in the renal pelvis, and one case of yellow granules in the common bile duct lumen.

Neuropathological findings:

not examined

Description (incidence and severity):

Not applicable.

Histopathological findings: non-neoplastic:

not examined

Description (incidence and severity):

Not applicable.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

Not applicable.

Other effects:

not examined

Description (incidence and severity):

Not applicable.

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:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Among 24 females mated in each group, 23, 24, 22 and 23 were confirmed to be pregnant in the 0 (control), 1, 30 and 1000 mg/kg bw/day groups. Live fetuses were obtained from all these pregnant females.

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Details on maternal toxic effects:

1000 mg/kg bw/day:
21 females exhibited soiled perigenital fur from Day 7 to day 20 of gestation (Day 2 of administration to the day of necropsy). An increased incidence of alopecia (nine cases) also suggested to be treatment-related since no such finding was seen in the control group. Significantly lowered values were observed in body weight gains from Days 0 - 10 of gestation to Days 0 - 20 of gestation (from Day 5 of dosing to the day of necropsy) and during dosing period (Days 6 - 20 of gestation). Significantly lowered values of food consumption during Days 6 - 9 of gestation (Days 1 - 4 of dosing) were observed.

30 mg/kg bw/day:
Significant reductions in maternal body weight gains were found during Days 0 - 10 and Days 0 - 12 of gestation (Day 5 and day 7 of dosing).

No significant difference was observed in gravid uterine weights, the number of corpora lutea and implantations, and the implantation index between treated and control groups.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: No adverse effects seen at this dose level.

Key result

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

1 mg/kg bw/day group:
Fetal body weights were not significantly different from that in the control group.

30 and 1000 mg/kg bw/day:
Male and female fetal weights were significantly lower than those in the control group.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

No significant difference was observed between the control and treated groups in the number of dead or resorbed embryos and fetuses, incidence of dead or resorbed embryos and fetuses, the number of live fetuses and fetal viability index.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Description (incidence and severity):

Not applicable.

Changes in postnatal survival:

not examined

Description (incidence and severity):

Not applicable.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were one or two cases of dwarf fetuses in each group. However, none of these fetuses had any other accompanying external malformations. No significant difference was observed between the control and treated groups in the fetal and litter incidences of external malformations.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Skeletal examinations of live fetuses revealed splitting of cervical vertebral body in a single fetus at 1000 mg/kg bw/day group. There was no significant difference between the control and treated groups in both fetal and litter incidences of skeletal malformation.

Skeletal variations were seen in all groups including the control group. The types and respective group mean incidences of skeletal variations calculated based on the fetal incidences per each litter in the 0 (control), 1, 30 and 1000 mg/kg groups were as follows: lumbar ribs, 37.70%, 41.67%, 61.23% and 46.46%; 14th ribs, 1.97%, 9.86%, 10.36% and 29.69%; and lumbarization of sacral vertebra, 1.24%, 5.78%, 6.41% and 5.51%. Other skeletal variations concordantly observed in the control and treated groups were cervical ribs in the control, 1 mg/kg and 1000 mg/kg groups at their respective incidences of 0.72%, 0.83% and 3.67% and unossification of thoracic vertebral body in the control, 30 mg/kg and 1000 mg/kg groups at their respective incidences of 2.17%, 0.65% and 0.62%. In addition, some fetuses in the control group exhibited wavy ribs (0.62%), spilitting of sternebra (0.62%) and asymmetry of sternebra (1.35%), fetuses in the 1 mg/kg group had splitting of ossification center of thoracic vertebral body (0.52%), dumbbell shape of ossification center of thoracic vertebral body (0.60%) and 7 lumbar vertebrae (0.52%), and fetuses in the 30 and 1000 mg/kg group showed unossification of lumbar vertebral body (0.65%) and wavy ribs (2.07%), respectively.

Among these skeletal variations, fetal incidences of lumbar ribs and fetal and litter incidences of lumbarization of sacral vertebra in the 30 mg/kg bw/day group were significantly higher than those in the control group. In the 1000 mg/kg bw/day group, both fetal and litter incidences of 14th ribs increased significantly. No further significant difference was seen between the control and treated groups in fetal and litter incidences of other skeletal variations.

Group mean incidences of fetuses with one or more skeletal variations calculated based on the total incidence of fetuses with skeletal variation(s) in each litter were significantly higher in the 30 and 1000 mg/kg bw/day groups when compared to the control group (41.01%, 52.05%, 71.59%, and 80.44% in the control, 1, 30 and 1000 mg/kg bw/day groups). In the 30 and 1000 mg/kg bw/day groups, the numbers of ossified cervical vertebral bodies, sacrocaudal vertebral bodies, total vertebral bodies and sternebrae decreased significantly. Significantly lowered values were also seen in the numbers of ossified metacarpal bones (right and total of both sides) and metatarsal bones (right side) in the 30 mg/kg bw/day group and in the number of ossified metatarsal bones (right, left, and total of both sides) in the 1000 mg/kg bw/day group. In addition, the number of ossified thoracic vertebral bodies increased significantly in the 1000 mg/kg bw/day group as a reflection of increased incidence of fetuses with 14th ribs.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no visceral malformations in any of the treated groups, including controls.

Visceral variations were observed in all groups including the controls. No significant difference was found in the incidences of fetuses or litters with visceral variations in the treated groups when they were compared to those in the control group (33.94%, 25.51%, 25.07%, and 33.56% in the control, 1, 30 and 1000 mg/kg bw/day groups).

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Details on embryotoxic / teratogenic effects:

Effects on the fetuses were limited to reduced fetal weights and an increase in skeletal variations, all of which occured at maternally toxic doses. None of the findings recorded in the fetuses were considered to be indicative of a teratogenic effects.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects seen at this dose level.

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

other: Increased number of skeletal variations

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: forelimb

skeletal: sternum

skeletal: rib

skeletal: supernumerary rib

skeletal: vertebra

skeletal: hindlimb

Description (incidence and severity):

Skeletal variations:
Significant increases in lumbarization of sacral vertebra at 30 mg/kg bw/day. Significant increase (both fetal and litter incidences) of 14th ribs at 1000 mg/kg bw/day.

Group mean incidence of skeletal variations, the numbers of ossified cervical vertebral bodies, sacrocaudal vertebral bodies, total vertebral bodies and sternebrae decreased significantly at 30 and 1000 mg/kg bw/day. Significant decrease in the numbers of ossified metacarpal bones (right and total of both sides) and metatarsal bones (right side) at 30 mg/kg bw/day and in the number of ossified metatarsal bones (right, left, and total of both sides) at 1000 mg/kg bw/day. Significant increase in the number of ossified thoracic vertebral bodies at 1000 mg/kg bw/day, as a reflection of increased incidence of fetuses with 14th ribs.

Developmental effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

The study was performed under GLP conditions and according to OECD 414. The test substance caused suppression of maternal body weight gain at 30 and 1000 mg/kg bw/day. Effects on maternal appearance and decreased food consumption were also observed at 1000 mg/kg bw/day. Test substance related suppression of fetal growth and increased incidence of fetal skeletal variations was observed at 30 and 1000 mg/kg bw/day. No increase was seen in the incidences of malformed fetuses at any dose level examined. Based on these results, the no-observed adverse effect level (NOAEL) was 1 mg/kg bw/day for both maternal animals and their fetuses under the conditions of the study, and it was concluded that the test substance is not teratogenic up to a dose level of 1000 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

0.1 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

Developmental toxicity was assessed in rodent (rat) and non-rodent (rabbit), according to OECD TG 414 (2001), under GLP conditions. The available information comprises an adequate and reliable study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-X, 8.7, of Regulation (EC) No 1907/2006.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Three developmental toxicity studies are available for the test substance. Two studies were conducted in rat and one in rabbit. All three studies were conducted in accordance with OECD TG 414 and under GLP conditions. A two-generation reproductive toxicity study conducted in accordance to OECD TG 416 and under GLP conditions is also available, in which the ability of the test substance to induce developmental toxicity was assessed.

Developmental Toxicity – Rat (first study: M-000336-01-1):

In this developmental toxicity study, sperm-positive Sprague-Dawley rats were exposed to the test substance by gavage from gestation day (GD) 6 to 20. The doses given were 0, 10, 80 and 600 mg/kg bw/day in suspension in aqueous solution of 0.5% methylcellulose 400. The sperm-positive females were allocated to groups (25 females per group); the sperm-positive day being GD 0. The volume of administration was 10 ml/kg bw based on the most recent body weight recorded.

Clinical observations were recorded daily. Maternal body weights were recorded for all the females on GD 0, 6, 8, 10, 12, 14, 16, 18 and 21. Food consumption was also measured for all the females during the intervals GD 0-6, 6-8, 8-10, 10-12, 12-14, 14-16, 16-18 and 18-21. At scheduled sacrifice, on GD21, the gravid uterine weight was recorded and the dams were evaluated for number of corpora lutea, number and status of implantations (resorptions, dead and live fetuses). The liver of all pregnant females was weighed and was examined microscopically at 600 mg/kg bw/day and in the control females. Live fetuses were removed from the uteri, counted, weighed, sexed and examined externally. Approximately half of the live fetuses from each litter were fixed in Bouin's solution and subsequently dissected for internal examination. The remaining half were eviscerated, fixed in absolute ethanol and stained according to a modification of the Tyl and Marr technique, for skeletal examination of bone and cartilage.

There were no mortalities during the course of the study.

600 mg/kg bw/day:

In dams, treatment-related clinical signs consisted of increased salivation observed in all females, increased incidence of hair loss, plus two females noted with yellow coloured staining around the anogenital region and an isolated incidence of liquid faeces. Body weight gain was markedly reduced by 69% between GD 6 to 8 and by 22% between GD 8 to 10 and GD 10 to 14, though during this latter interval, the effect was accentuated by the marked loss in body weight of one female which was found to have a torn oesophagus, considered to have occurred during dosing on GD 10. Food consumption was reduced by 9% between GD 6 to 8 and by 6% between GD 8 to 12.

Maternal corrected body weight change was 11% lower than in the control group. Liver weight was increased by 16% at this dosage. At the macroscopic examination one female had a liver with prominent lobulation. Microscopic examination of the liver at this dosage revealed no treatment related changes.

Mean fetal body weights (calculated by sex and combined sex) were reduced by 10-11%, the effect being statistically significant for both separate and combined sexes. There was a slight increase in the mean percentage of runt fetuses at this dosage. At the visceral examination, there was an increase in the incidence of dilated ureters (a variant), in comparison with the control group. At the skeletal examination, two fetuses were noted with malformations, one concerned poor ossification of the sacrocaudal vertebrae and the absence of cartilaginous sacrocaudal vertebrae.

The second malformation concerned changes to the cervical vertebrae and costal cartilages. In isolation, these findings are considered to be incidental and not treatment-related. The following anomalies were clearly elevated at 600 mg/kg bw/day; short 14th rib associated with the presence of 27 pre-sacral vertebrae, bipartite/dumbbell thoracic centrum and cartilage, and unossified hyoid centrum. Treatment-related variants consisted of poor ossification in the 7th cervical centrum, several different sternebrae together with bipartite and misaligned 5th sternebra and misaligned 4th sternebra, 3rd and 4th proximal phalanges of the forepaw, sacrocaudal vertebrae, 5th metacarpal, 1st metatarsal and 1st thoracic centrum. In addition, increased incidence of extra ossification points on the 14th vertebra, short 14th rib and 27 presacral vertebrae were also noted.

80 mg/kg bw/day:

In dams, treatment-related clinical signs consisted of increased salivation observed in 2/25 females and increased incidence of hair loss. Body weight gain was markedly reduced by 66% and food consumption reduced by 11%, between GD 6 to 8. Material corrected body weight change was 11% lower than in the control group.

Mean fetal body weights (calculated by sex and combined sex) were reduced by 4%, the effect being statistically significant for both separate and combined sexes.

At the visceral examination, there was an increase in the incidence of dilated ureters (a variant) at this dosage. At the skeletal examination poor ossification was noted in the 7th cervical centrum, 5th and 6th sternebrae together with an increased incidence of bipartite and misaligned 5th sternebra, 3rd and 4th proximal phalanges of the forepaw, and sacrocaudal vertebrae. In addition, there was an increase in the incidence of extra ossification points on the 4th vertebra and short 14th rib. These findings are classified as variants.

10 mg/kg bw/day:

No maternal toxicity was observed at 10 mg/kg bw/day. In terms of developmental toxicity, at the visceral examination, there was an increase in the incidence of dilated ureters (a variant). At the skeletal examination, poor ossification was observed in the 7th cervical centrum, 5th and 6th sternebrae together with an increased incidence of bipartite and misaligned 5th sternebra, and extra ossification points on the 14th vertebra. These findings are classified as variants.

In conclusion, the test substance when administered by oral gavage to the pregnant rat caused slight maternal toxicity and slight retardation in fetal maturation at both 600 and 80 mg/kg bw/day. A dose level of 10 mg/kg bw/day was found to be the NOEL in terms of maternal toxicity, whilst producing a very slight retardation in fetal maturation, the toxicological significance of which is considered to be doubtful. A dose level of 10 mg/kg bw/day was therefore considered to be the NOAEL in terms of developmental toxicity.

Developmental Toxicity – Rat (second study: M-400385-01-2):

In this developmental toxicity study, the test substance was administered daily by oral gavage to copulated BrlHan:WIST@Jcl (GALAS) female rats (n=24 per group) from Day 6 to 19 of gestation. The doses given were 0, 1, 30 and 1000 mg/kg bw/day in suspension in an aqueous solution of 0.5% methylcellulose. The volume of administration was 10 mL/kg bw based on the most recent body weight recorded. The sperm-positive females were allocated to groups; the sperm-positive day being gestation Day 0.

No deaths occurred during the study period. Clinical signs included soiled perigenital fur and alopecia at 1000 mg/kg bw/day. Maternal body weight gain was significantly reduced at Days 0-10 and Days 0-12 of gestation (Day 5 and 7 of administration) at 30 mg/kg bw/day. At 1000 mg/kg bw/day, significantly lowered values were seen in maternal body weight gain from Day 5 of dosing to the day of necropsy (from Days 0-10 of gestation to Days 0-20 of gestation) and during dosing period (Days 6-20 of gestation), as well as in food consumption of dams during Days 6-9 of gestation (Days 1-4 of dosing). Observations of the ovaries and uterine contents at necropsy on gestation Day 20 revealed no changes related to the test substance on the gravid uterine weight, numbers of corpus lutea and implantations, implantation index, number of dead or resorbed embryos and fetuses, incidence of dead or resorbed embryos and fetuses, number of live fetuses, viability index of fetuses, fetal sex ratio, and placental weights in any dosing groups.

Fetal body weights were significantly lower at 30 and 1000 mg/kg bw/day when compared to the controls.

No treatment related findings were observed during both the external and visceral examinations.

No treatment related skeletal malformations were observed. No treatment-related changes were found in the incidences of skeletal variations in the 1 mg/kg bw/day group. At 30 mg/kg bw/day there was a significant increase in the incidence of fetuses with lumbar ribs and those of fetuses and litters with lumbarization of sacral vertebrae. At 1000 mg/kg bw/day there was a significant increase in incidences of fetuses and litters with 14th ribs. This led to a significant increase in the incidence of fetuses with one or more skeletal variations at 30 and 1000 mg/kg bw/day. Among skeletal variations described above, both lumbar ribs and 14th ribs are indicative of extra rib formation but they were merely classified based on whether they were accompanied by the presence of extra vertebrae, suggesting the occurrence of a cascade of skeletal variations. Thus, the present results can be interpreted that test substance treatment induced the formation of extra ribs at 30 and 1000 mg/kg bw/day, which was followed by an increase in vertebral number at 1000 mg/kg bw/day. The increases in these variations are thought to be related to the test substance administration because a significant increase or similar tendency was also noted in the fetal incidences of lumbarization sacral vertebrae and 14th ribs at 100 and 1000 mg/kg bw/day in a dose range-finding study. Fetal ossification was delayed at 30 and 1000 mg/kg bw/day, in which the numbers of ossified bones in the cervical vertebral bodies, sacral/coccygeal vertebral bodies, total vertebral bodies, sternebrae, and metacarpal bones or metatarsal bones decreased significantly. These reductions in number of ossified bones are thought to be associated with lowered fetal body weights. In addition, the number of ossified thoracic vertebral bodies increased significantly at 1000 mg/kg bw/day as a reflection of increased incidence of fetuses with 14th ribs.

Under the conditions of the study, the NOAEL for maternal and fetal effects was 1 mg/kg bw/day, based on suppression of maternal food consumption and body weight gain, and suppression of fetal growth and increased incidence of fetal skeletal variations. No increase was seen in the incidences of malformed fetuses at any dose levels examined, therefore it was concluded that the test substance is not teratogenic up to the dose level of 1000 mg/kg bw/day.

Since the NOAEL for developmental toxicity at the low dose of 10 mg/kg bw/day was equivocal in the first study (M-000336-01-1), and a clear NOAEL for developmental toxicity has been established at a lower dose of 1 mg/kg bw/day on the more recently conducted study (M-400385-01-2), on which a wider dose range was evaluated, then the first study is considered to be superseded by the more recent study. Consequently, the data on the first rat study are only discussed within this endpoint summary, and no study record was included in the technical dossier.

Developmental Toxicity – Rabbit (M-398566-01-2):

In a developmental toxicity study, the test substance was administered daily by oral gavage to artificially inseminated Japanese White rabbits (n=25 per group) from Day 6 to 27 of gestation. The doses given were 0, 0.1, 10 and 1000 mg/kg bw/day in suspension in an aqueous solution of 0.5% methylcellulose. The volume of administration was 5 mL/kg bw based on the most recent body weight recorded.

No maternal toxicity was noted at 0.1 and 10 mg/kg bw/day. At 1000 mg/kg bw/day, six animals (including two non-pregnant animals) died during Days 20 to 28 of gestation. These animals showed decreases in food consumption, or had stopped feeding, and decreases in body weight before death. Five animals (including two non-pregnant animals) exhibited clinical findings as soiled fur and red discharge on the tray. One of the animals aborted on the day of death. In addition, the mean body weight gains on Day 15 of gestation and thereafter and the mean food consumption during Days 6 to 9 and Days 15 to 18 of gestation were significantly lower in this dose group than those in the control group.

No fetal toxicity was noted in the 0.1 mg/kg group. At 10 and 1000 mg/kg bw/day, body weights of male fetuses were significantly lower than those in the control group and the incidences of supernumerary rib and 27 presacral vertebrae as skeletal variations increased markedly at skeletal observations. In addition, the incidences of fetuses with any skeletal variations in the 10 and 1000 mg/kg bw/day groups were significantly higher than that in the control group due to the increased incidences of these skeletal variations. The numbers of live fetuses, the incidences of resorptions and fetal death and the results of other teratological examinations in the 10 and 1000 mg/kg bw/day groups were not influenced by the treatment.

Under the conditions of the study, the NOAEL for maternal effects was 10 mg/kg bw/day, based on reduced body weight gain and food consumption, adverse clinical findings and mortality in maternal animals. The NOAEL for fetal effects was 0.1 mg/kg bw/day, based on reduced fetal weights in males and increased skeletal variations in males and females at 10 and 1000 mg/kg bw/day. No severe effects like structural malformations or embryo/fetal lethality were observed. Further, the incidence of fetal malformations in the 1000 mg/kg bw/day group was not elevated compared to that which induced extra ribs (10 mg/kg bw/day). Taking all these aspects together, it was concluded that the test substance did not show teratogenic effects in rabbits under the conditions of the study. Overall, based on the type and severity of fetal effects, the results obtained in the rabbit are not considered to support classification concerning developmental toxicity.

Two-generation reproductive toxicity study (M-399182-01-2):

The test substance was mixed in the basal feed at 0 (basal feed), 2, 20, and 200 ppm and given to 24 male and 24 female SPF Wistar Hannover rats/ dose group for two successive generations to evaluate the potential effects on reproductive performance and on the growth and development of their offspring. The study was performed under GLP and according to OECD 416.

Effects of the test substance treatment on pups were noted at a dose level of 20 ppm and above, in which body weight gains of pups were reduced significantly and dose-dependently. Opacity of the eyeballs was induced in pups of the 200 ppm group at and after postnatal day 21, although its incidence was low. Histopathological observations also revealed keratitis in some weanlings of the 20 and 200 ppm groups, of which incidences in F1 male weanlings in the 20 ppm group and in F1 and F2 male and female weanlings in the 200 ppm group were significantly higher than those in the control group. However, these findings are not considered to be toxicologically relevant to humans but rather species specific. No toxicologically relevant findings were noted in pups of the 2 ppm group.

The necropsy of weanlings showed the absolute weights of the brain, spleen and thymus in F1 and F2 male and female weanlings in the 20 ppm and higher dose groups often showed significant low values. However, the relative weights of these organs were similar to or significantly higher than those in the control group, being considered to be the changes caused by low body weights. Furthermore, the relative weight of the brain in F2 male weanlings in the 2 ppm group was significantly low, whereas there were no significant differences in the 20 ppm and higher dose groups, being considered to be an accidental change. Furthermore, an increase in liver weight, in which the differences from controls were statistically significant in relative liver weights of F1 weanling in the 20 and 200 ppm groups and those of F2 weanlings in all the treated groups. Thus, all the weighed livers were examined histopathological. However, the examinations did not show any changes related to the test substance treatment except for focal necrosis of the hepatocyte in 1 to 2 weanling of the treated group, suggesting that an increase in liver weights of weanlings was an adaptive response. Dilatation of the renal pelvis was noted in F1 and F2 weanlings in each group including the control group and there was a statistically significant difference of the incidence of this finding in F2 weanlings between the 200 ppm group and the control group. There was no tendency of increasing incidence of this finding in the 2 and 20 ppm groups in each generation.

In the observation of sexual development in F1 parental male animals, the age of completion of preputial separation was delayed statistically significantly at 20 ppm and above. However, these changes are considered to be due to lowered body weights that were caused by the test substance treatment. In addition, measurements of anogenital distance did not reveal the influence of test substance treatment in the absolute and relative values of F2 pups.

Treatment-related effects were only noted in offspring at the same dose levels at which maternal toxicity was observed. Based on the reduced body weights at 20 ppm, the no observed adverse effect level of the test substance in offspring was concluded to be 2 ppm. Based on dietary intake of the parental animals, this corresponds to: P1 generation 0.126 mg/kg bw/day (males) and 0.202 mg/kg bw/day (females); F1 generation 0.142 mg/kg bw/day (males) and 0.204 mg/kg bw/day (females).

Supplementary Study: Effects of tyrosinaemia on pregnancy and embryo-fetal development in the rat (M-263626-01-3):

The objectives of this study were to assess the relationship between increase in blood tyrosine level and potential effects on pregnancy and embryo-fetal development. The study was conducted under GLP and according to the OECD guideline 414 (2001), with the exception of visceral fetal examinations.

Tyrosinaemia was induced by a co-administration to sperm-positive females Sprague-Dawley rats of 2-(2-Nitro-4-trifluoromethylbenzoyl) cyclohexane-1,3-dione(NTBC), an inhibitor of 4-hydroxyphenylpyruvic acid dioxygenase (4-HPPDase), at 10 µg/kg/day by gavage from gestation day (GD) 6 to 20 and L-Tyrosine at 2% (w/w) in the diet from GD 6 to 21. Similar groups were co-administered vehicle alone (demineralized water) by gavage plus untreated diet (control group), or NTBC by gavage plus untreated diet, or vehicle alone by gavage plus diet supplemented with L-Tyrosine at the same dosage. The sperm-positive females were allocated to four groups (23 females per group); the sperm-positive day being GD 0. The volume of administration of NTBC preparation was 10 ml/kg bw based on the most recent body weight recorded.

Clinical observations were recorded daily. A detailed physical examination including careful examination of the eyes was conducted on GD 1, 8, 15 and 21. Maternal body weights were recorded for all females on GD 0, 6, 8, 10, 12, 14, 16, 18 and 21. Food consumption was also measured for all the females during the intervals GD 1-6, 6-8, 8-10, 10-12, 12-14, 14-16, 16-18 and 18-21. At scheduled sacrifice, on GD 21, blood samples were collected for determination of L-Tyrosine level. Liver was weighed fresh for pregnant females. Liver, thyroid gland and pancreas from all females were preserved in 10% neutral buffered formalin, whilst eyes were preserved in Davidson's fixative for possible histological examination. The gravid uterine weight was recorded and the dams were evaluated for number of corpora lutea, number and status of implantations (resorptions, dead and live fetuses). Live fetuses were removed from the uteri, counted, weighed, sexed and examined externally. All live fetuses were eviscerated, fixed in absolute ethanol and stained according to a modification of the Tyl and Marr technique, for skeletal examination of bone and cartilage on approximately half of the live fetuses from each litter.

There were no treatment-related mortalities or clinical signs during the course of the study. The pregnancy rate was unaffected by treatment with L-Tyrosine at 2% in the diet, or oral administration of NTBC at 10 µg/kg/day, or co-administration of L-Tyrosine at 2% in the diet plus NTBC at 10 µg/kg/day by gavage.

L-Tyrosine at 2% (group 2)

In dams, mean body weight gains, mean body weights, mean corrected body weight change and mean food consumption were unaffected by treatment. At the macroscopic examination, mottled kidney (bilateral) was noted in 1/23 females. Mean maternal liver weight was unaffected by treatment. Blood tyrosine level at autopsy was increased by a factor of 4.7, compared to the control group. Hence, administration of diet containing 2% w/w L-Tyrosine between GD 6 to 21 (equivalent to 1433 mg/kg/day) provoked a limited tyrosinaemia, in comparison to the control group.

At caesarean section, uterine content examination did not reveal any treatment-related effects on any litter parameter assessed, including fetal body weights, number of live fetuses, early and late resorptions, fetal death status and percentage of male fetuses.

No treatment-related findings were noted at the external fetal and fetal skeletal examinations.

NTBC at 10 µg/kg bw/day (group 3)

One female was sacrificed prematurely on GD 13 after reduced motor activity and tilting head were observed on GD 12 and 13. This female lost 21 g in body weight between GD 10 and 12, together with reduced food consumption, compared with the control group. At autopsy, no macroscopic changes were noted. No clear relationship to the treatment could be established.

In dams, mean body weight gain was reduced by 58% between GD 6 to 8 in comparison to the controls. Thereafter, mean body weight gains were comparable to the control values, resulting in an overall body weight gain between GD 6 to 21 similar to the control group. Mean corrected body weight change and mean food consumption were unaffected by treatment. At the macroscopic examination, mottled kidney (bilateral) was noted in 2/23 females. Mean maternal liver weight was unaffected by treatment. Blood tyrosine level at autopsy was increased by a factor of 8.4, compared to the control group. Hence, oral gavage administration of NTBC at 10 µg/kg bw/day between GD 6 to 20 provoked a slight tyrosinaemia, in comparison to the control group.

At caesarean section, the only litter parameter affected was mean fetal body weight, where there was a slight 2% reduction at both the combined and separate sex level.

No treatment-related findings were noted at the external fetal and fetal skeletal examinations.

L-Tyrosine at 2% + NTBC at 10 µg/kg bw/day (group 4)

In dams, mean body weight gain was reduced by 55% between GD 6 to 8 in comparison to the controls. Thereafter, mean body weight gains were similar to the control values, but the overall body weight gain between GD 6 to 21 was still slightly reduced by 6% in comparison to the control group. Mean corrected body weight change was unaffected by treatment, whilst mean food consumption was slightly decreased by 8% between GD 18 to 21, compared to the control group. At the macroscopic examination, mottled kidney (bilateral) was noted in 3/23 females and minimal corneal opacity (unilateral or bilateral) was noted in 4/23 animals. Mean maternal liver weight was unaffected by treatment. Blood tyrosine level at autopsy was increased by a factor of 63, compared to the control group. Hence, administration of diet containing 2% w/w L-Tyrosine between GD 6 to 21 (equivalent to 1424 mg/kg bw/day) plus oral gavage administration of NTBC at 10 µg/kg bw/day between GD 6 to 20 provoked a marked tyrosinaemia, in comparison to the control group.

At caesarean section, the only litter parameter affected was mean fetal body weight, where there was a 7% reduction at both the combined and separate sex level, the effect being statistically significant in each case.

No treatment-related findings were noted at the external fetal examination. At the fetal skeletal examination, an increase in the incidence of no or incomplete ossification was noted for the 7th cervical centrum, 5th and 6th sternebrae, 3rd and/or 4th proximal phalanges of forepaws, 5th metacarpal, 1st metatarsal and sacrocaudal vertebrae, in comparison to the controls. In addition, higher incidences than in the control group were noted for short 14th rib and extra ossification points on the 14th thoracic vertebra. All these findings were classified as variant findings.

In conclusion, co-administration of L-Tyrosine at 2% w/w in the diet between GD 6 to 21 plus NTBC by oral gavage at 10 µg/kg/day between GD 6 to 20 provoked a marked maternal tyrosinaemia which caused a general delay of ossification in fetuses.

The findings from this supplementary study correlate with the findings seen within the reproductive and developmental toxicity studies conducted with the test substance, where delayed fetal maturation was observed, which was predominantly characterized by reduced body weights and delays in ossification. Thus, the findings of delayed fetal maturation can be attributed to increases in maternal plasma tyrosine levels caused by the test substances ability to inhibit of 4-hydroxyphenylpyruvic acid dioxygenase (4-HPPDase), an enzyme of the tyrosine catabolic pathway. This common mode of action for 4-HPPDase inhibitors is discussed in a US EPA review paper (M-808741-01-1), where it is demonstrated that developmental effects are consistent across the respective 4-HPPDase inhibiting substances, these effects are primarily characterized by decreased body weights, skeletal variations and delays in skeletal ossification. Thus, as increases in plasma tyrosine levels following exposure to 4-HPPDase inhibitors are less pronounced in humans than in rats and rabbits, as humans have a greater capacity to clear excess tyrosine from their system through alternative pathways (as discussed in the repeated dose toxicity endpoint summary), then the aforementioned findings in the developmental studies in rats and rabbits for the registered substance are not considered to be relevant to humans.

 

Overall conclusion on developmental toxicity

In utero exposure to the test substance led to overall fetal effects including depressed body weight and the increase in skeletal variations in rats and rabbits. Further, delayed ossification was observed in rats. No severe effects like structural malformations or embryo/fetal lethality were observed in the conducted developmental toxicity studies, neither in rats nor in rabbits. Overall, based on the available data, it was concluded that the test substance did not show teratogenic effects. Based on the type and severity of the observed fetal effects, the results obtained in rats and rabbits are not considered to support classification concerning developmental toxicity.

Mode of Action Analysis / Human Relevance Framework

Please refer to the information provided in the repeated dose toxicity and toxicity to reproduction endpoint summaries.

Justification for classification or non-classification

The available two-generation reproductivity toxicity study with 2-{2-chloro-4-mesyl-3-[(tetrahydrofuran-2-ylmethoxy)methyl]benzoyl}cyclohexane-1,3-dione (CAS 473278-76-1) meets the criteria for classification according to Regulation (EC) No 1272/2008 regarding systemic toxicity, and is therefore classified as STOT RE Cat. 1 (H372).

Regarding reproductive performance, the test substance does not meet the criteria for classification according to Regulation (EC) No 1272/2008, and is therefore conclusive but not sufficient for classification.

In the developmental toxicity studies, no findings were seen that were indicative of teratogenic activity. In addition, the developmental effects seen in the two-generation reproductive toxicity study occurred only under systemic toxic effects of parental animals and therefore are considered to be secondary. It is concluded that the test substance does not meet the criteria for classification according to Regulation (EC) No 1272/2008, and is therefore conclusive but not sufficient for classification.