Ethyl [2-(4-phenoxyphenoxy)ethyl]carbamate

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

- Oral: NOAEL for systemic toxicity = 200 ppm (mean dietary equivalent to 14 and 17 mg/kg bw/day for males and females, respectively; NOAEL for reproductive toxicity > 1800 ppm (mean dietary equivalent to 127 and 146 mg/kg bw/day for males and females, respectively); NOAEL for developmental toxicity = 600 ppm (mean dietary equivalent to 42 and 49 mg/kg bw/day for males and females), respectively, feeding study, rat, EPA 83.2, Barker and Goodyer 1986

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8 Sep 1983 to 14 Dec 1984

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 83-4 (Reproduction and Fertility Effects)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Albino (Crl:CD(SD)BR)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: (P) 6 weeks; (F1) 3.5 to 6.5 weeks
- Weight at study initiation: (P) Males: 200 to 285 g; Females: 144 to 201 g; (F1) Males: 132 to 226 g; Females: 106 to 178 g
- Housing: The animals were housed in groups or individually according to the phase of the study. Group housed animals were caged in stainless steel wire mesh cages suspended above aluminium trays with cardboard liners. Individually housed females and dames with litters were caged in solid floor polypropylene cages with stainless steel grid tops. Autoclaved sawdust was provided for bedding and, during parturition, shredded paper was provided as nesting material
- Diet: Powdered Rat and Mouse diet ad libitum
- Water: Ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 40 to 70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 8 Sep 1983 to 14 Dec 1984

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: Separate batches of diet were prepared for each treatment group at weekly intervals throughout the study. Any diet remaining at the end of each week was discarded
- Storage temperature of food: Room temperature

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 21 days
- Proof of pregnancy: Sperm in vaginal smear referred to as day 0 of pregnancy
- After 10 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged: The females were housed individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Batches of diet were prepared at concentrations corresponding to the proposed low and high dose levels. Quintuplicate samples (each from different levels of the mix) were taken from each batch and analysed in duplicate for their test substance content, to provide an indication of homogeneity.
The batches of diet were then stored at room temperature and triplicate samples were removed from each batch 7 and 14 days after preparation. The samples were then analysed to investigate the stability of the test article at room temperature.
The concentration of the test substance in was determined in duplicate samples of each experimental diet during weeks 1, 12, 25, 37, 49 and 61 to confirm the accuracy of the preparation

Duration of treatment / exposure:

For the F0 animals, this was from initiation until the end of the F0-F1b mating period (F0 males) or until all the F1b litters were weaned (F0 females). For the selected F1b animals, this was from the time they began eating independently until the end of the F1-F2b mating period (F1 males) or until all the F2b litters were weaned (F1 females)

Frequency of treatment:

Continuously

Details on study schedule:

- Selection of parents from F1 generation when pups were 7 days of age.
- Age at mating of the mated animals in the study: F0: 80 days, F1: 100 days

Dose / conc.:

200 ppm

Remarks:

Low dose: Dietary equivalent to 14 and 17 mg/kg bw/day for males and females, respectively

Dose / conc.:

600 ppm

Remarks:

Mid dose: Dietary equivalent to 42 and 49 mg/kg bw/day for males and females, respectively

Dose / conc.:

1 800 ppm

Remarks:

High dose: Dietary equivalent to 127 and 146 mg/kg bw/day for males and females, respectively

No. of animals per sex per dose:

F0: 30
F1: 25

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dietary concentrations were selected by the study sponsor after examination of data from a 6 week dose range-finding study in the rat.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
- Time schedule: At the beginning and end of each working day

GENERAL CLINICAL OBSERVATIONS:
- Time schedule: Daily. Towards the end of gestation, pregnant females were examined twice daily for signs of parturition
- Clinical observations: Signs of ill health, toxicity or behavioural change

BODY WEIGHT:
- Time schedule for examinations: Weekly until the confirmation of mating. Thereafter, body weights were recorded on days 0, 6, 12, 15 and 20 of gestation and on days 1,7, 14 and 21 of lactation. Following lactation, body weights were recorded weekly.

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, at weekly intervals during the pre-mating periods only

OTHER:
- In the event of a delay between discovery of a dead animal and its necropsy, the carcass was stored at approximately 4 °C to minimise tissue autolysis
- The following data were recorded: date of mating, date of parturition and duration of gestation.

Oestrous cyclicity (parental animals):

Not examined

Sperm parameters (parental animals):

Not examined

Litter observations:

PARAMETERS EXAMINED
The following data were recorded for each litter:
- Number of pups born (live and dead)
- Number of pups alive on days 1, 4, 7, 14 and 21 post-partum
- Individual pup weights on days 1, 4, 7, 14 and 21 post-partum
- Sexes of pups alive on days 1, 4, 7 and 21 post-partum general condition of pups throughout the pre-weaning period (birth to day 21 post-partum).

On day 4 post-partum litters were culled to a maximum of eight pups (4 male+ 4 female, where possible). Pups were selected by random card draw. Any pup dying during lactation was necropsied, where possible, to investigate the cause of death. Culled pups were also necropsied.

The following developmental parameters were measured for each litter:
- Pinna unfolding,
- Fur and hair growth,
- Tooth eruption
- Eye opening.

For each parameter, the number of pups in each litter showing the observation on each day was recorded until all the pups in the litter had shown the observation. In addition, on day 21 post-partum (weaning), 2 male and 2 female pups per litter (where possible) were selected for the following functional tests:
- Grip strength
- Pupillary reflex (both eyes)
- Visual placing response
- Auditory response.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after the second mating
- Maternal animals: All surviving animals after 21 days of weaning.

GROSS NECROPSY
Females: The following tissues and organs were taken from each animal and preserved in 10% neutral buffered formalin: uterus vagina*, ovaries*, target organ (liver)*.
Males: The following tissues were prepared for macroscopic examination and weighed: Testes*, epididymides, seminal vesicles, prostate and target organ (liver)*.
(*) organs were weighed prior to fixation

HISTOPATHOLOGY / ORGAN WEIGHTS
- Histopathological examinations were performed on the tissues and organs of all control and high dose males.
- Histopathological examination was performed on the tissues and organs of all control and high dose females. The uteri of pregnant females, killed or found dead, were examined. Implantations were subdivided into: Normal for date foetuses and resorbed foetuses

OTHER:
Twenty five days after the second pairing period in each generation, females that failed to litter were killed and necropsied. The uterus was examined for evidence of implantation before fixation.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days of age.

GROSS NECROPSY
- All F1a, F2a and the non-selected F1b pups were subjected to macroscopic examination. The following tissues and organs were taken from one male and one female pup per litter (where possible) and preserved in 10% neutral buffered formalin: *adrenals, *brain (fore-, mid-, hind-), caecum, colon duodenum, +eyes (both), femur (including bone marrow), *heart, ileum, jejunum, *kidneys, *liver (2 lobes), lung (2 sections, coronal cut), mesenteric lymph node, ovaries/uterus testes/epididymides/prostate/seminal vesicles pancreas, pituitary, salivary glands (mandibular), spinal cord (cervical and lumbar), spleen, stomach, thymus, thyroids, trachea, urinary bladder, vagina and all gross lesions
(*) organs weighed before fixation

- All F2b offspring were subject to macroscopic examination. From one male and one female pup per litter (where possible), the following tissues and organs were taken and preserved in 10% neutral buffered formalin: #*adrenals, #*brain, caecum, colon, #duodenum, #+eyes (both), femur (including bone marrow), *heart, ileum, jejunum, #*kidneys, #*liver (2 lobes), #lung (2 sections, coronal cut), #mesenteric lymph node, #ovaries/uterus, #testes/epididymides/prostate/seminal vesicles, #pancreas, #pituitary, salivary glands (mandibular), spinal cord, (cervical and lumbar), #spleen, #stomach, #thymus, #thyroids, trachea, #urinary bladder, #vagina and all gross lesions (*) organs weighed before fixation
(+) preserved in Davidson's fluid.
(#) Histopathological examination of all organs was performed on the control and high dose pups.

HISTOPATHOLOGY / ORGAN WEIGTHS
Organs and tissues for histopathological examination were embedded in paraffin wax BP (mp 56°C), sectioned at a nominal thickness of 5 microns and stained with haematoxylin and eosin.
- F1b pups: histological examination was performed only on the livers of 5 male and 5 female F1b pups form each of groups 1 and 4.
- F2b pups: histopathological examination of all organs marked with an # in the list above was performed on the control and high dose pups.

Statistics:

Continuous or semi-continuous responses and some discrete responses: Statistical evaluation was made using an analysis of variance technique for normally distributed errors on by non-parametric techniques for non-normally distributed errors. Analysis of variance established the significance of 'the variability between all groups to determine a treatment-related response. The standard deviation obtained from this analysis was used for 't'-tests between the control and treatment groups. Where necessary, the data were suitably transformed before analysis. Non-parametric testing was carried out using the Kruskal-Wallis test to determine a treatment-related response. Significant differences between control and treatment groups were determined using the Wilcoxon rank sum test. All tests were carried out at li and 5% significance levels for a two sided risk.
Discrete responses: Statistical analysis was carried out using Fisher's two-sum randomisation (permutation) test with a Monte Carlo simulation for computation of significance levels. The litter was the experimental unit and a square root transformation was used for weighting the number of incidences and adjusting the
different litter sizes. Each treatment group was tested against the control at 1% and 5% significance levels for a one-sided risk. In the interpretation of statistical analyses p values greater than 0.05 were considered to be not significant

Reproductive indices:

Mating index, fertility index and the fecundity index

Offspring viability indices:

Gestation index, live birth index, viability index 1 (day 1 to 4), viability index 2 (day 4 to 7), viability index 3 (day 7 to 14), viability index 4 (day 14 to 21) and pre-weaning loss (day 0 to 21).

Clinical signs:

no effects observed

Description (incidence and severity):

There were no abnormalities of clinical condition considered to be related to treatment. Non-specific changes of the type commonly seen in reproduction studies with this strain of rat occurred with similar frequency across all groups.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One group 4 male, died during week 17. One group 2 female was killed in extremis on day 7 of gestation in the F1a littering phase of the study.
During the second mating phase, at the time of parturition, a number of females exhibited dystocia - often with languor and tremors. Some animals died, some were killed in extremis and some were able to litter, but the pups were born dead or died shortly after birth. Five group 1 females died or were killed on days 21 or 22 of gestation, two group 2 females and one group 4 female were killed on day 21. In addition, one female from each of groups 2 and 4 died on day 1 of lactation. The pups died on day 1 from 2 further litters, one from group 2 and one from group 4. The problem at parturition appeared to be associated with severe stress as a consequence of unusually large litter sizes. All the females that died or were killed had litter sizes larger than the norm. The incidence of affected females was not dose-related.
All mortalities were considered to be incidental to treatment.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

During the pre-mating period, the mean body weight gain for the treated males and females was comparable to or higher than the control in groups 2 and 3 and marginally lower in group 4. The group 4 weight gain was significantly lower than controls in females for weeks 0 to 4 only.
For the remainder of the study, including (for females) both gestation and lactation phases, weight gains were comparable to or higher than controls for both sexes in all groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

During the pre-mating period the food consumption of the parental animals was generally comparable between all groups, and although marginally lower values were observed for group 4 females, no statistically significant intergroup differences were observed.

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Microscopically, treatment-related changes were seen in the liver of some of the group 4 parental animals, minimal to slight hypertrophy was recorded in 19 out of 30 parental· males and 13 out of 30 females; moderate periportal hypertrophy was seen in 1 male. In addition, minimal to slight focal necrosis was seen in 5 out of 30 group 4 males and one female compared to 2 out of 30 control males. The severity was moderate to marked for the control animals. A further group 4 male had severe lobar necrosis. Two group 4 females had moderate to marked centrilobular necrosis, which was the same incidence and similar severity to the control females. There was no evidence of a treatment-related effect on any other tissue. examined.
- No treatment-related changes were seen at necropsy for the selected F1 a or F1 b pups. Histological examination of the livers of the group 4 F1 b pups revealed no abnormalities.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

effects observed, treatment-related

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

- All males mated during the F0-F1a mating period and only 2 males (one from each of groups 2 and 4) failed to mate during the F0-F1b mating period.
- All females mated, at both pairings, generally within one oestrous cycle. After the first pairing (F1a) only 2 animals, one group 1 and one group 3 were not pregnant. After the second pairing (F1b) 7 animals were not pregnant, 2 from each of groups 1, 2 and 3 and one from group 4. Only one group 3 animal failed to conceive after both pairings.
- There was no effect of treatment on litter size for either the F1a or F1b litters. The mean number of pups born per dam in the treated groups was similar to or larger than the control mean and there was little intergroup variation in sex ratio

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

200 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Remarks on result:

other: Dietary equivalent to 14 and 17 mg/kg bw/day for males and females, respectively

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity

Effect level:

> 1 800 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Dietary equivalent to 127 and 146 mg/kg bw/day for males and females, respectively

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no abnormalities of clinical condition considered to be related to treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One group 3 male was killed in extremis during week 24. There were no deaths in the females on the F2a littering phase of the study. As was seen in the first (F0) generation a number of females showed dystocia at the second (F2 b) littering phase and died or were killed. Four group 1 females died between day 22 of gestation and day 1 of lactation; 2 group 2 females died on day 22 of gestation; one group 3 female was killed on day 21 of gestation and one group 4 female died on day 1 of lactation. It was considered that the F1 females were past their peak of reproductive performance at the time of the second littering phase and probably this contributed to the observed difficulties in parturition.
All mortalities were considered to be incidental to treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- The group 4 parental animals were slightly lower in body weight than the controls at the start of the F1 generation. The mean weight of the males was 9% lower than the control weight and the female weight was 5% lower. The rate of body weight gain for group 4 was generally similar to the controls, although a significantly lower weight gain was observed in females from weeks 8 to 12. The initial body weights and the weight gain in groups 2 and 3 were similar to the controls.
- The initial body weights and the weight gain in groups 2 and 3 were similar to the controls.
- The body weight gain for treated females during the gestation and lactation periods of both the F2a and F2b pregnancies were comparable to the control gain.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

During the pre-mating period the food consumption of the parental animals was similar in groups 1, 2 and 3 and marginally lower in group 4. The reduction was statistically significant in group 4 females for weeks 1 to 5 only.

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- The mean relative liver weights of group 3 females and group 4 parental males and females were significantly increased when compared to the control (increase: group 3 female 19%; group 4 male 15%, female 36%). The mean relative weight for other parental animals was similar to the control.
- There was no treatment-related change in the relative weights of the other weighed organs from the parental animals.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopically, treatment-related changes were seen in the liver of some of the group 4 parental animals. Minimal to moderate hypertrophy was seen in 14 out of 25 group 4 females. In addition, minimal to slight focal necrosis was seen in 14 out of 25 group 4 males; the incidence of focal necrosis for the females was similar to that of the controls. There was no evidence of a treatment-related effect on any other tissue examined.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

- There was no effect of treatment at any dose level on mating performance or fertility.
- Compared to the F0 generation, there was an increase in the number of males that did not mate twice in the F1 generation. However, there was no conclusive association with treatment as the incidence was 5, 3, 5 and 4 in groups 1 to 4, respectively. Also, only three of these animals (2 group 3 and 1 group 4 ) failed to mate on both occasions.
- All females mated at both pairings, generally within 1 oestrous cycle. The pregnancy incidence in the treated groups was higher than the control group at both pairings.
- The mean duration of gestation was comparable to controls in group 2, marginally shorter in group 3 and significantly reduced in group 4. In the F2b littering phase significant reductions in the duration of gestation were observed in groups 3 and 4. Several animals also showed difficulties in parturition, but the incidence of affected females was not dose-related.
- Only 2 females, one from each of groups 1 and 2 failed to conceive after both pairings.
- There was no effect of treatment on litter size. The mean number of pups born per dam in the treated groups was similar to or higher than in the control group. Sex ratios were within the normal range in all groups

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

200 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

food consumption and compound intake

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Remarks on result:

other: Dietary equivalent to 14 and 17 mg/kg bw/day for males and females, respectively

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity

Effect level:

> 1 800 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Dietary equivalent to 127 and 146 mg/kg bw/day for males and females, respectively

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There was no effect of treatment of the clinical condition.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Survival of the offspring was unaffected by treatment. However, the pre-weaning loss for group 2 in the F1b littering phase was higher than normal. This was due to 3 females showing total litter loss during the first 2 days post-partum.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- For the F1 a litters the mean pup weight at day 1 post-partum was similar in all groups. However, subsequently the mean weight of the control pups increased at a marginally greater rate than in the treated groups. The difference in weight was significant in all groups by day 14 and at weaning on day 21 post-partum the mean pup weight increase for groups 2, 3 and 4 was 92, 93 and 89% of the control weight increase, respectively.
- For the F1 b litter the mean pup weight at day 1 post-partum was similar in all groups. Subsequently the mean weight of groups 2 and 3 pups increased at a similar rate to the controls. The mean weight of the group 4 pups was again significantly lower than the control by day 14. At weaning on day 21 post-partum the mean weight increases for group 2, 3 and 4 were 98.1, 95.6 and 89.1% of the control weight increase, respectively.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- The mean relative liver weight of the group 4 F1a female pups killed on day 21 post-partum was marginally increased, by 5%, when compared to the control.
- At the F1b kill the mean relative liver weight for group 4 male and female and group 3 female pups were significantly increased, compared to the control (increase: group 3 female 10%; group 4 male 19%; female 19%). Group 3 male relative liver weight were also slightly increased (5% compared to control) but the difference was not statistically significant.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No necropsy findings were found on the offspring, dead or culled pups from either of the F1 litters.

Histopathological findings:

no effects observed

Description (incidence and severity):

Histological examination of the livers of the group 4 F1b pups revealed no abnormalities.

Other effects:

no effects observed

Description (incidence and severity):

There was very little intergroup difference in the physical development of the offspring, in either of the F1 litters, as assessed by the intra litter onset and duration of pinna unfolding, hair growth, tooth eruption and eye opening. Functional development of the offspring of both F1 litters was unaffected by treatment.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental

Generation:

F1

Effect level:

600 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: Dietary equivalent to 42 and 49 mg/kg bw/day for males and females, respectively

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There was no effect of treatment on the clinical condition of the offspring.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Survival of the offspring was unaffected by treatment. Pre-weaning losses in the treated groups were similar to or lower than in the control group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- In both littering phases (F2 a and b) the mean pup weights at day 1 and day 4 post-partum were slightly lower in the treated groups than the control weights. However, the control litter size was slightly smaller than in the treated groups and it was considered that the slightly heavier control pups were due to reduced intra-litter competition resulting from the smaller litter size. When this bias was eliminated (by standardisation of litter size at day 4) weight gain in groups 2 and 3 was comparable to controls and only marginal reductions were observed in group 4.
- Significant differences in pup weight were, however, observed in the treated groups as weight gain after day 4 did not compensate for the early retardation.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- The mean relative liver weights of the group 4 F2a pups killed on day 21 post-partum was significantly increased (by 5 to 6%) when compared to the control. The mean relative weight for other pups and F2a pups was similar to the control. At the F2 b kill the mean relative liver weight of the group 4 females was only marginally increased (by 4%) when compared to the control. The mean relative liver weights of the other F2b pups were comparable to the control weight, although some significant differences were observed in absolute weights.
- Statistically significant differences in relative brain weight were observed in group 4 male F2a pups and all treated female F2b selected pups, but were considered to be associated with the retardation of body weight seen in these pups rather than a direct effect of treatment.
- Significant increases in relative kidney weight were observed in treated male F2b selected pups only and were considered incidental to treatment as there was no effect of the F2b female selected pups or the F2a selected pups.
- There was no treatment-related change in the relative weights of the other weighed organs from the selected F2a or F2b offspring.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment related changes were seen at necropsy for the selected F2a and F2b pups.

Histopathological findings:

no effects observed

Description (incidence and severity):

Histological examinations of the livers of the group 4 F2b pups revealed no abnormalities.

Other effects:

no effects observed

Description (incidence and severity):

The physical and functional development of the offspring of both F2 litters was unaffected by treatment.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental

Generation:

F2

Effect level:

600 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

Remarks on result:

other: Dietary equivalent to 42 and 49 mg/kg bw/day for males and females, respectively

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Results analysis of test diet formulations:

The homogeneity of the test substance in the low dose and the high dose diet mixes was considered to be acceptable (mean values of 101.0 and 100.2% of nominal). In addition, the stability of the test substance in the diet was also considered to be acceptable (7% loss/week at 250 µg/g). The homogeneity and stability tests were completed before dosing started.

The concentration of the test substance in diets analysed during the F0 generation phase of the study were close to the nominal concentrations (95.5 - 102.5% of nominal) and were considered to be acceptable.

The concentration of the test article in diets analysed during the F1 generation phase of the study were close to the nominal concentrations (97.5 to 105% of nominal) and were considered to be acceptable.

Table 1. Group reproduction indices F0 -F1a

Parameter	Group 1	Group 2	Group 3	Group 4
Total number of females	30	30	30	30
Total number of mated females	30	30	30	30
% females mating	100	100	100	100
Mating index %	87.9	90.9	100	93,8
Total number of pregnant females	29	30	29	30
Fertility index %	96.7	100	96.7	100
Fecundity index %	96.7	100	96,7	100
Gestation index %	100	96.7	100	100
Live birth index %	96.4	95.5	96.6	97.6
Viability index 1 %	90.8	86.8	90.2	93.5
Viability index 2 %	99.7	99.1	100	100
Viability index 3 %	100	99.7	100	99.5
Viability index 4 %	100	100	99.7	100
Pre- weaning loss %	12.6	18.1	13.1	9.2

Table 2. Group reproduction indices F0 -F1b

Parameter	Group 1	Group 2	Group 3	Group 4
Total number of females	30	29	30	30
Total number of mated females	30	29	30	30
% females mating	100.0	100.0	100.0	100.0
Mating index %	96.6	93.1	100.6	100.0
Total number of pregnant females	28	27	28	29
Fertility index %	93.3	93.1	93.3	96.7
Fecundity index %	93.3	93.1	93.3	96.7
Gestation index %	82.1	88.9	100	93.1
Live birth index %	96.5	89.l	97.4	92.2
Viability index 1 %	91.3	90.0	90.S	95.2
Viability index 2 %	99.3	99.3	99.4	100.0
Viability index 3 %	99.6	99.3	99.4	98,7
Viability index 4 %	100.0	100.0	100.0	100.0
Pre- weaning loss %	12.9	21.0.	12.9	13.3

Table 3. Group mean litter data, pup number F1a

Parameter	Group 1	Group 2	Group 3	Group 4
Number of pregnancies	29/30	30/30	29/30	30/30
% of pregnancies	96.7	100.0	96.7	100.0
Mean duration of gestation (days)	21.s	21.4	21.3	21.3-
Number of pups born	419	419	413	424
Mean number per dam	14.4	14.4	14.2	14.1
Sex ratio males : females	1:1.14	1:0.91	1:1.00	1:1.03
Number of pups alive day 1	404	400	399	414
Mean number per dam	13.9	13.8	13.8	13.8
Number of pups alive day 4	367	347	360	387
Mean number per dam	12.7	12	12.4	12.9
Number of pups culled day 4	143	131	136	147
Mean number per dam	4.9	4.5	4.7	4.9
Number of pups alive day 7	223	213	224	240
Mean number per dam	7.7	7.3	7.7	8
Number of pups alive day 14	223	212	224	238
Mean number per dam	7.7	7.3	7.7	7.9
Number of pups alive day 21	223	212	223	238
Mean number per dam	7.7	7.3	7.7	7.9
% pre-weaning loss	12.6	18.1	13.1	9.2

Table 4. Group mean litter data, pup number F1b

Parameter	Group 1	Group 2	Group 3	Group 4
Number of pregnancies	28/30	27/29	28/30	29/30
% of pregnancies	93.3	93.1	93.3	96.7
Mean duration of gestation (days)	23	24	28	27
Mean duration of gestation (days)	21.7	21.3	21.3*	21.2**
Number of pups born	311·	338	389	361
Mean number per dam	13.5	14.1	13.9	13.4
Sex ratio males : females	1:1.19	1:0.99	1:0.95	1:1.02
Number of pups alive day 1	300	301	379	333
Mean number per dam	13.0	12.5	13.5	12.3
Number of pups alive day 4	274	271	343	317
Mean number per dam	11.9	11.3	12.3	11.7
Number of pups culled day 4	97	114	134	118
Mean number per dam	4.2	4,8	4.8	4,4
Number of pups alive day 7	175	155	207	199
Mean number per dam	7.6	6,S	7.4	7.4
Number of pups alive day 14	174	153	20S	19S
Mean number per dam	7.6	6,4	7.3	7.2
Number of pups alive day 21	174	153	205	195
Mean number per dam	7.6	6.4	7.3	7.2
% pre-weaning loss	12.9	21.0	12,9	13.3

Statistical analysis: Intergroup differences from control group duration of gestation statistically significant: p<0,05*   p<0.01**

Conclusions:

The NOAEL is set at 200 ppm for systemic parental toxicity (mean dietary equivalent to 14 and 17 mg/kg bw/day for males and females, respectively, the NOAEL for reproductive toxicity is set at 1800 ppm (mean dietary equivalent to 127 and 146 mg/kg bw/day, respectively and the NOAEL for developmental toxicity is set at  600 ppm (mean dietary equivalent to 42 and 49 mg/kg bw/day for males and females.

Executive summary:

A two-generation reproduction study of the test substance was conducted in the Sprague Dawley-derived rat of the Crl:CD(SD)BR strain according to EPA 83.2 and GLP principles. Groups of 30 male and 30 female (F0 generation) or 25 male and 25 female (F1 generation) rats were given the test substance orally, in the diet, at concentrations of 200, 600 or 1800 ppm (groups 2, 3 and 4, respectively). Similar groups of rats given untreated diet acted as controls (group 1). For males the dietary equivalent were 14, 42 and 127 mg/kg bw/day and for females 17, 49 and 146 mg/kg bw/day for males and females, respectively. For 600 ppm, the dietary intakes were equivalent to mg/kg bw/day. After a period of maturation (F0: 80 days, F1: 100 days) the parental animals were mated (1M:1F) and the females were allowed to rear their offspring to weaning (F0-F1a; F1-F2a). One week after weaning was completed, the parental animals were paired and allowed to rear their offspring to weaning for a second time (F0-F1b; F1-F2b). The animals which formed the F1 generation were randomly selected from the F1b litters. In each generation, the parental animals were evaluated for treatment-related effects on survival, clinical condition, body weight gain, food consumption, reproductive performance and pathological changes. The offspring were evaluated for effects on viability, growth, clinical condition, physical and functional development and pathological changes. The concentration of test article in the formulated diets which were analysed during weeks 1, 12, 25, 37, 49 and 61 was close to the nominal concentration and was considered acceptable for the purpose of this study. The homogeneity and stability of the test substance in the diet were assessed and considered acceptable before the start of the study.

Results showed that there were no treatment-related mortalities of the parental animals in either the F0 or F1 generations. In both generations, several females died during or shortly after parturition of the second litters. These deaths were attributed to the animals being past their peak of reproductive performance and to their unusually large litter sizes and were considered incidental to treatment. There were no abnormalities of clinical condition in the parental animals of either generation which could be considered treatment-related. During the pre-mating period, body weight gain of the F0 parental animals was comparable to controls in groups 2 and 3 but marginally lower in group 4. For the remainder of the F0 treatment period, body weight gain was comparable to controls in all groups. There was no effect of treatment at any dose level on body weight gain of the F1 parental animals although the group 4 animals were slightly lighter at the start of treatment. Food consumption during the pre-mating period was marginally lower than controls in group 4 females during the F0 generation and in group 4 males and females during the F1 generation. There was no effect of treatment in groups 2 and 3 on food consumption during either the F0 or F1 generation. Mating performance and fertility were unaffected by treatment at any of the dose levels investigated at either the first or second mating in each generation. Slight reductions in the duration of gestation were observed in groups 3 and 4 in the second mating of the first generation and both matings in the second generation. There was no effect of treatment in either generation on the number of pups born, neonatal viability, or clinical condition or necropsy findings of the offspring arising from either mating phase. A marginal reduction in the rate of weight gain was observed in both group 4 litters in each of the F0 and F1 generations. Neonatal growth in groups 2 and 3 was comparable to controls in each littering phase. The physical and functional development of the offspring of all treated animals was similar to controls in both litters of each generation. All group 4 parental animals showed a statistically significant increase in both absolute and relative liver weight, with marginal increases also noted in group 3 females. In general, group 4 offspring also showed increased liver weight relative to body weight, although the effect was less distinct than in the adults. Gonad weights were slightly increased in group 4 F0 adults only, although there were no adverse effects on reproductive performance. Treatment-related liver changes (hypertrophy/focal necrosis) were observed in the majority of the group 4 F0 or F1 parental animals. When compared to controls, both the incidence and severity of these findings were increased at this dose level. There was no evidence of a treatment-related effect on any other tissue examined, or on the livers of the selected F1b or F2b pups.

In conclusion, treatment at 1800 ppm elicited slight toxicity in the parental animals, characterised by marginal, temporary effects on weight gain, food intake and on gonad weight, liver weight and liver pathology. There were no adverse effects at this dose level on clinical condition, reproductive performance, or on neonatal viability, condition, development or pathology. However, minimal effects on pup growth and lover weight were observed in each generation. At 600 ppm, slight increases in liver weight were observed only in the parental females (F1) and female F1b pups. A slight reduction in the duration of gestation was also observed in each generation. No other adverse effects were observed at this dose level. There was no effect of treatment at 200 ppm on either the parental animals or their offspring. Therefore the NOAEL is set at 200 ppm for systemic parental toxicity (mean dietary equivalent to 14 and 17 mg/kg bw/day for males and females, respectively, the NOAEL for reproductive toxicity is set at 1800 ppm (mean dietary equivalent to 127 and 146 mg/kg bw/day, respectively and the NOAEL for developmental toxicity is set at  600 ppm (mean dietary equivalent to 42 and 49 mg/kg bw/day for males and females.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

127 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Guideline study (EPA 83.2) performed in compliance with GLP

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Toxicity to reproduction, Barker and Goodyer 1986

A two-generation reproduction study of the test substance was conducted in the Sprague Dawley-derived rat of the Crl:CD(SD)BR strain according to EPA 83.2 following GLP principles. Groups of 30 male and 30 female (F0 generation) or 25 male and 25 female (F1 generation) rats were given the test substance orally, in the diet, at concentrations of 200, 600 or 1800 ppm (groups 2, 3 and 4, respectively). Similar groups of rats given untreated diet acted as controls (group 1). For males the dietary equivalent were 14, 42 and 127 mg/kg bw/day and for females 17, 49 and 146 mg/kg bw/day for males and females, respectively. For 600 ppm, the dietary intakes were equivalent to mg/kg bw/day. After a period of maturation (F0: 80 days, F1: 100 days) the parental animals were mated (1M:1F) and the females were allowed to rear their offspring to weaning (F0-F1a; F1-F2a). One week after weaning was completed, the parental animals were paired and allowed to rear their offspring to weaning for a second time (F0-F1b; F1-F2b). The animals which formed the F1 generation were randomly selected from the F1b litters. In each generation, the parental animals were evaluated for treatment-related effects on survival, clinical condition, body weight gain, food consumption, reproductive performance and pathological changes. The offspring were evaluated for effects on viability, growth, clinical condition, physical and functional development and pathological changes. The concentration of test article in the formulated diets which were analysed during weeks 1, 12, 25, 37, 49 and 61 was close to the nominal concentration and was considered acceptable for the purpose of this study. The homogeneity and stability of the test substance in the diet were assessed and considered acceptable before the start of the study.

Results showed that there were no treatment-related mortalities of the parental animals in either the F0 or F1 generations. In both generations, several females died during or shortly after parturition of the second litters. These deaths were attributed to the animals being past their peak of reproductive performance and to their unusually large litter sizes and were considered incidental to treatment. There were no abnormalities of clinical condition in the parental animals of either generation which could be considered treatment-related. During the pre-mating period, body weight gain of the F0 parental animals was comparable to controls in groups 2 and 3 but marginally lower in group 4. For the remainder of the F0 treatment period, body weight gain was comparable to controls in all groups. There was no effect of treatment at any dose level on body weight gain of the F1 parental animals although the group 4 animals were slightly lighter at the start of treatment. Food consumption during the pre-mating period was marginally lower than controls in group 4 females during the F0 generation and in group 4 males and females during the F1 generation. There was no effect of treatment in groups 2 and 3 on food consumption during either the F0 or F1 generation. Mating performance and fertility were unaffected by treatment at any of the dose levels investigated at either the first or second mating in each generation. Slight reductions in the duration of gestation were observed in groups 3 and 4 in the second mating of the first generation and both matings in the second generation. There was no effect of treatment in either generation on the number of pups born, neonatal viability, or clinical condition or necropsy findings of the offspring arising from either mating phase. A marginal reduction in the rate of weight gain was observed in both group 4 litters in each of the F0 and F1 generations. Neonatal growth in groups 2 and 3 was comparable to controls in each littering phase. The physical and functional development of the offspring of all treated animals was similar to controls in both litters of each generation. All group 4 parental animals showed a statistically significant increase in both absolute and relative liver weight, with marginal increases also noted in group 3 females. In general, group 4 offspring also showed increased liver weight relative to body weight, although the effect was less distinct than in the adults. Gonad weights were slightly increased in group 4 F0 adults only, although there were no adverse effects on reproductive performance. Treatment-related liver changes (hypertrophy/focal necrosis) were observed in the majority of the group 4 F0 or F1 parental animals. When compared to controls, both the incidence and severity of these findings were increased at this dose level. There was no evidence of a treatment-related effect on any other tissue examined, or on the livers of the selected F1b or F2b pups.

In conclusion, treatment at 1800 ppm elicited slight toxicity in the parental animals, characterised by marginal, temporary effects on weight gain, food intake and on gonad weight, liver weight and liver pathology. There were no adverse effects at this dose level on clinical condition, reproductive performance, or on neonatal viability, condition, development or pathology. However, minimal effects on pup growth and lover weight were observed in each generation. At 600 ppm, slight increases in liver weight were observed only in the parental females (F1) and female F1b pups. A slight reduction in the duration of gestation was also observed in each generation. No other adverse effects were observed at this dose level. There was no effect of treatment at 200 ppm on either the parental animals or their offspring. Therefore the NOAEL is set at 200 ppm for systemic parental toxicity (mean dietary equivalent to 14 and 17 mg/kg bw/day for males and females, respectively, the NOAEL for reproductive toxicity is set at 1800 ppm (mean dietary equivalent to 127 and 146 mg/kg bw/day, respectively and the NOAEL for developmental toxicity is set at  600 ppm (mean dietary equivalent to 42 and 49 mg/kg bw/day for males and females.

Effects on developmental toxicity

Description of key information

- Oral: NOAEL for maternal toxicity is 100 mg/kg bw/day; the NOAEL for developmental toxicity is 300 mg/kg bw/day, rat, OECD 414, Hummler and McKinney 1984

- Oral: NOAEL for maternal toxicity is 50 mg/kg bw/day, the NOAEL for developmental toxicity is 500 mg/kg bw/day, rat, OECD 414, Eckhardt 1983

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Aug 1982 to 24 Sep 1982 (Experiment A), 18 Jul 1983 to 19 Aug 1983 (Experiment B)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

1981 and 2001

Qualifier:

according to guideline

Guideline:

other: Food and Drug Administration. Guidelines for Reproduction Studies for Safety Evaluation of Drugs for Human Use

Version / remarks:

1966

Qualifier:

according to guideline

Guideline:

other: Committee on Safety of Medicines. Guidelines on Reproduction Studies for Guidance of Applicants for Product Licences and Clinical Trial Certificates, June.

Version / remarks:

1974

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rabbit

Strain:

other: Swiss Hare

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: Experiment A, 2440-3500 g and experiment B, 2250-3530 g
- Housing: The animals were individually housed in stainless steel cages
- Diet: Diet cubes ad libitum
- Water: Tap water ad libitum
- Acclimation period: Minimum of 4 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18
- Humidity (%): 50
- Air changes: Fully air- conditioned
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 23 Aug 1982 to 24 Sep 1982 (Experiment A), 18 Jul 1983 to 19 Aug 1983 (Experiment B)

Route of administration:

oral: gavage

Vehicle:

other: SSV (4% carboxymethylcellulose, 0.9% NaCl, 0.5% benzylalcohol, 0.4% Tween 80 in water)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was formulated freshly each week in the vehicle. High shear mixing was used during formulation and the suspension was kept homogeneous during dosing by the use of a magneto stirrer

VEHICLE
- Amount of vehicle: 5 mL/kg

Analytical verification of doses or concentrations:

yes

Details on mating procedure:

- Impregnation procedure: Cohoused
- M/F ratio per cage: 1:1
- Proof of pregnancy: sperm in vaginal smear referred to as day 1 of pregnancy

Duration of treatment / exposure:

From day 7 to 19 inclusive of gestation

Frequency of treatment:

Once daily

Duration of test:

Until day 30 of gestation

Dose / conc.:

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

Remarks:

Experiment A, group II

Dose / conc.:

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

Remarks:

Experiment A, group III

Dose / conc.:

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

Remarks:

Experiment A, group IV

Dose / conc.:

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

Remarks:

Experiment B, group II

No. of animals per sex per dose:

Experiment A: 20 mated females
Experiment B: 35 mated females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In a range finding study for maternal sensitivity and embryotoxicity, the substance doses of 100 and 300 mg/kg bw/day were administered to pregnant rabbits. Based on these results, dosages of 30, 100 and 300 mg/kg bw/day were selected for the principle study
- Foetuses from a necropsy subgroup were removed by ovariohysterectomy one day before parturition and processed for skeletal and visceral examinations. Additionally, dams from a rearing subgroup were allowed to deliver naturally and to raise their young until weaning. All females with litters were necropsied after the 23rd day of lactation and the uteri were examined for implantation sites. The offspring was examined externally.
- Accurate interpretation of the findings from the principle study was increasingly complicated by the appearance of some malformations mainly in the 300 mg/kg dose group which rendered inconclusive results. Therefore, a supplementary study was conducted with increased numbers of females using a dose of 200 mg/kg in an attempt to reproduce the same effects and to establish the relevance of the malformations that arose in the 300 mg/kg group. The same treatment schedule was used as in the principle study and included a separate control group.

Maternal examinations:

CAGE SIDE OBSERVATIONS:
- Time schedule: Daily, all changes in behaviour, general condition, signs of pharmacological action, etc., rabbits which during the experiment were autopsied

BODY WEIGHT:
- Time schedule for examinations: On day of gestation 1, 7, 20 and 30

Ovaries and uterine content:

The ovaries and uterine content was examined after termination:
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

Fetal examinations:

- External examinations: Yes, all per litter
- Soft tissue examinations: Yes, all per litter
- Skeletal examinations: Yes, all per litter
- All the young were tested for their 24h viability in an incubator at 34 °C

Statistics:

1. Descriptive statistics:
- In the tables for each group, median and interquartile- range are indicated. The median has the property, that 50 % of all values in the group are smaller and 50 % are larger than the median. The interquartile range contains 50 % of the values in the group, excluding the 25 % smallest and the 25 % largest values.
Exp. A: Statistical evaluations employed the T-TEST for independent samples and/or the CHI—SQUARE—TEST. The data presented in the tables are calculated mean values and standard deviations.
2. Significance tests:
- Trend set: All groups together are tested by a simultaneous test against trend (Jonckheere-test) or Armitage-test as indicated in the table. In case of a significant result, the correspondent interpretation (‘’*’’ for 5 % > p ≥ 1 % and ‘’**’’ for p < 1 % is attached to the highest dosage group. The test is then repeated without the already "significant" highest dosage group and again interpreted as before.
- Simultaneous test without ordered alternative: In the case of a non-significant result of the trend test, the same (remaining) groups are tested by a simultaneous test without trend alternative (Kruskal-Wallis-test and Chi^2-test as indicated in the table). If this test again is not significant (p= 1%) the test procedure ends: all remaining groups are not significantly different from control. P is chosen as 1% for this test to keep the total error probability near 5%
- Two sample tests for each dosage group against control: In the case of a significant result for test b), all dosages groups are separately tested against control by means of a two sample test (U-test or Fisher-test as indicated) the interpretations mean: ‘’#’’: 5% > p ≥ 1% and ‘’##’’: p<1%
3. Experimental unit: The dam is considered the independent experimental unit within all significant tests
4. Relative numbers: (relative number = absolute number/number of implantations)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two dams at 100 mg/kg bw/d died on day 12 and 19 (cause of death unknown).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain was reduced in dams at 300 mg/kg bw/d during the treatment period (80% of control) and at 200 mg/kg bw/d from the treatment period till necropsy (76-90% of control).

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

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

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental

Effect level:

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

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal examination of the neonates by radiography and/or Alizarin Red staining revealed no compound- related effects on any of the measured ossification parameters and all findings from the treatment group of both the principle and supplementary studies compared favorably to the respective controls.

Visceral malformations:

no effects observed

Description (incidence and severity):

The examination for soft tissue abnormalities revealed no indication of any drug-related deviation in any dose group

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

In the principle study (A) macroscopic examination of the foetuses at necropsy from the control group revealed one foetus with a hypoplastic tail. A dose of 30 mg/kg revealed one foetus with a hypoplastic tail and two additional litter mates with tails missing. From a dose of 100 mg/kg there were two foetuses with missing tails, one foetus with an open eye and one foetus with spina bifida. The malformations at the highest dose of 300 mg/kg included two cases of spina bifida with hypoplastic tails, one single case from both spina bifida and open eyes, a single foetal resorption with omphalocele and two cases of hypoplastic tail. From the supplementary study (B) with 200 mg/kg the only observed malformation was localized in a foetal resorption which had open eyes and a reduction malformation of the right arm. Contrary to results in study A, the control group of study B contained malformations in which the type and incidence is
analogous to those found in the 300 mg/kg dose group. These included two foetuses with ectopy; one of which had a missing tail, one foetus with an eye open, a foetus with omphalocele and a foetus with hypoplastic tail. Although the malformations arising from study. A were considered as being questionably compound-related, further investigation has undoubtedly shown that an association with the test substance can be excluded. That the observed malformations in study A are not compound- related is mainly the result of substantiating evidence collected from the supplementary study (B). It was assumed that a reproducible pattern of the same types of malformations would have appeared in the 200 mg/kg if those seen in the 300 mg/kg group were actually relevant. But neither pattern nor associated malformations Were established and no relationship exist in the results between the two dose groups. Furthermore, though the incidence of malformations of increased in the 300 mg/kg dose group the same type of malformations are often observed among historical controls but in particular among the controls in the supplementary study. In addition there were also no malformations noted among the foetuses in the preliminary study at doses of 100 and 300 mg/kg. Therefore, in view of the various supporting
data, all of the observed malformations originating in the principle study are considered only to be arbitrary findings

Key result

Dose descriptor:

NOAEL

Effect level:

>= 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Table 1. Results experiment A

Dose (mg/kg bw/day)	0	30	100	300	dr
Maternal effects Mortality (n=20) Clinical signs Pregnant animals Body weight gain Food consumption Uterus weight Pathology macroscopy	0   18	0            2 No treatment-related findings 20 17   Not performed Not determined   Not performed		0   20   dc
Litter response
Number of dams	18	20	17	20
examined
Corpora lutea/dam		No treatment-related findings
Total number of resorptions/dam	2.2	2.1	1.2	2.8
Number of resorptions/group
- embryonic	38	41	14	50
- foetal	1	2	6	5
Dams with live foetuses	17	17	17	18
Live foetuses/dam	6.8	5.0	6.4	6.3
Dead foetuses	0	0	0	0
Foetal weight	41.8	44.3	44.6	43.6
Post-implantation loss/dam^1	2.1	2.2	1.2	2.7

Dose (mg/kg bw/day)	0	30	100	300	dr
Sex ratio	No treatment-related findings
Mean crown-rump length	No treatment-related findings
Survival rate 24 h	No treatment-related findings
Examination of the foetuses	122   0   1 (0.8%)	101 109   0 1 (0.9%) 1 0 (1.0%) No treatment-related findings No treatment-related findings		126   3 (2.4%) 4 (3.2%)
Total no foetuses
External observations - spina bifida, sacral region - hypoplastic tail
Skeletal findings
Visceral findings

dr: dose related

dc/ic: statistically significantly decreased/increased compared to the controls

d/i: decreased/increased, but not statistically significantly compared to the controls

a/r: absolute/relative organ weight

1: as calculated by the reviewer; no statistical analysis performed

Table 2. Results experiment B

Dose (mg/kg bw/day)	0		200		dr
Maternal effects Mortality Clinical signs Pregnant animals Body weight gain Food consumption Uterus weight Pathology macroscopy	35	None  No treatment-related findings   Not performed Not determined   Not performed		33   d
Litter response
Number of dams examined	35			33
Corpora lutea/dam		No treatment-related findings
Total number of resorptions/dam	1.0			1.0

Dose (mg/kg bw/day)	0		200		dr
Number of resorptions/group - embryonic - foetal	34 11		28 3
Dams with live foetuses	35		33
Live foetuses/dam	7.0		8.0
Foetal weight	42.4		41.6
Post-implantation loss/dam^1	2.0		1.0
Sex ratio	No treatment-related findings
Mean crown-rump length	No treatment-related findings
Survival rate 24 h	No treatment-related findings
Examination of the foetuses	349   0   1 (0.3%)	No treatment-related findings No treatment-related findings		234   0   0
Total no foetuses
External observations - spina bifida, sacralregion - hypoplastic tail
Skeletal findings
Visceral findings

Table 3. Historical control data from Hoffmann LaRoche, between 1977 and 1988

	Control Gronp		Foetuses with Finding
	N of Litters	N of foetuses	hypoplasfictail	missing tail	open eyes	spina bifida	ompha /ocoele
TOTAL	539	3656	3	2	2	2	2
% of litters affected (range)			0-13.3	0-6.7	0-5.3	0-5.6	0-6.7
% of fetuses affected (range)			0-2.3	0-1.1	0-1.0	0-0.8	0-1.1

Conclusions:

Based on decreased body weight, the NOAEL for maternal toxicity was set at 100 mg/kg bw/day. Based on no effects on foetal development the NOAEL for developmental toxicity was set at ≥ 300 mg/kg bw/day.

Executive summary:

The test substance was tested for embryotoxic and teratogenic action in 20 mated (principle study) and 35 (supplementary study) female Swiss hare rabbits according to OECD TG 414 and GLP principles. In the principle study, doses of 30, 100 and 300 mg/kg bw/day were administered by oral gavage to pregnant animals from day 7 to 19 inclusive of gestation. A control group received the vehicle (SSV, 4% carboxymethylcellulose, 0.9% NaCl, 0.5% benzylalcohol, 0.4% Tween 80 in water) for the same treatment period. All females were sacrificed at day 30 of gestation. Foetuses were removed by ovariohysterectomy tested for viability (24 h) and examined for macroscopic, skeletal and soft tissue anomalies. Accurate interpretation of the findings of the principle study was increasingly complicated by the appearance of some malformations in the 300 mg/kg dose group which rendered inconclusive results. Therefore, a supplementary study was conducted with increased number of females using a dose of 200 mg/kg bw/day in an attempt to reproduce the same effects and to establish the relevance of the malformations that occurred in the 300 mg/kg group. The same treatment schedule was used as in the principle study and included a separate control group. Throughout the summary the principle and supplementary study will be referred as A and B.

Results showed a moderately reduced body weight development during the treatment period among females of the 200 and 300 mg/kg dose groups, but not a highly significant effect. There were no detectable effects on the measured reproductive parameters, on the course and outcome of pregnancy nor on the 24 hour survivability of the neonates in either study. In the principle study (A) beginning with a dose of 100 mg/kg, some morphological malformations appeared mainly as open eyes, spina bifida and/or short or missing tail. The incidence was low and compared favourably to the frequency of these malformations in observed historical control data of this rabbit strain. In spite of no effects seen in the preliminary study at a dose of 300 mg/kg, malformations did appear at this dose level in the principle study. The malformations were similar to those observed in the 100 mg/kg group but the incidence was somewhat higher than recognized in historical controls thus, complicating an accurate interpretation of the results. In the supplementary study (B) using a dose of 200 mg/kg, it was assumed that a pattern of the same types of malformations would appear if the effects seen in the 300 mg/kg group were relevant. However, the results from this study show that no pattern or associated malformation was established between the two doses and only one malformation was present and localized in a foetal resorption. Furthermore, in contrast to the single malformation from the control group of the principle study (A), the malformations seen in the control group of the study supplementary study (B) appeared more frequently and were analogous in both type and incidence to the malformations seen in the 300 mg/kg dose group. Therefore, in view of all substantiating evidence with particular emphasis on the high incidence of malformations seen in the controls of the supplementary study, all malformations seen in the principle study (A) of the 300 mg/kg group are considered to be arbitrary findings and not test substance-related. Examination of the foetuses for skeletal and soft tissue anomalies did not reveal any effects considered to be test substance-related from any dose group.

In conclusion, based on decreased body weight, the NOAEL for maternal toxicity was set at 100 mg/kg bw/day. Based on no effects on foetal development the NOAEL for developmental toxicity was set at ≥ 300 mg/kg bw/day.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 Jul 1982 to 19 Aug 1982

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

1981 and 2001

Qualifier:

according to guideline

Guideline:

other: Food and Drug Administration. Guidelines for Reproduction studies for Safety Evaluation of Drugs for Human Use

Version / remarks:

1966

Qualifier:

according to guideline

Guideline:

other: Committee on Safety of Medicines. Guidelines on Reproduction Studies for Guidance Applicants of for Product Licences and Clinical Trial Certificates

Version / remarks:

June 1974

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Fu-albino

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 210.8 ± 10.6 g to 215.1 ± 7.5 g
- Housing: 2 animals in wire mesh during acclimatization and mating. Females of rearing groups are caged individually in Macrolon cages with dust free wood shavings from day 21 during gestation and during lactation
- Diet: Diet cubes, ad libitum
- Water: Tap water in drinking bottles, ad libitum
- Acclimation period: Minimum of 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 50
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 30 Jul 1982 to 19 Aug 1982

Route of administration:

oral: gavage

Vehicle:

other: SSV (Standard Solvent Vehicle: 4% carboxylmethylcellulose, 0.9% NaCl, 0.5% benzylalcohol, 0.4% Tween 80 in H2O dest.)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was formulated once per 10 days of treatment in the vehicle and stored in a refrigerator. High shear mixing was used during formulation and the suspensions was kept homogeneous during dosing by the use of a magneto stirrer.

VEHICLE
- Concentration in vehicle: 5, 15 and 50 mg/mL
- Amount of vehicle: 10 mL/kg

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- M/F ratio per cage: 1/1
- Further matings after two unsuccessful attempts: Yes, the mating procedure was repeated until enough mated females were available
- Proof of pregnancy: Vaginal plug referred to as day 1 of pregnancy

Duration of treatment / exposure:

From day 7 to day 16 inclusive of gestation

Frequency of treatment:

Daily

Duration of test:

Until day 21 of gestation

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

36 mated female rats

Control animals:

yes

Details on study design:

- Dose selection rationale: Dose levels were based on a teratogenic range-finding study with rats exposed to 500 and 1000 mg/kg bw/day.

Maternal examinations:

CAGE SIDE OBSERVATIONS:
- Time schedule: Daily

BODY WEIGHT:
- Time schedule for examinations: At the beginning of the experiment, then daily during the treatment period, on day 17 and day 21 of gestation

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day 21, the dams of the necropsy sub-group were killed by inhaled carbon dioxide

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

Fetal examinations:

- Necropsy sub group 1: The uteri were examined for the number and location of implantations and resorptions. The uteri of females without foetuses were examined for implantations after staining according to the method of Salewski. The number of corpora lutea was determined. The foetuses were examined macroscopically and weighed. One half of the foetuses of 15 litters per dose group were eviscerated, macerated, stained with Alizarin Red S, cleared and preserved in glycerine for subsequent examination for skeletal anomalies. The second half of the 15 litters were fixed and subsequently examined for visceral abnormalities.
- Rearing sub- group 2: The dams of the rearing sub-group were allowed to litter spontaneously and to rear their young up to weaning. On the 1st, 4th, 12th and 23rd day of lactation litter size was registered and the young and the mothers were weighed. At the 23rd day after birth all offspring were examined externally (teeth, eyes, ears, quality of the fur, extremities, toes, external sex organs, anus, tail) killed and discarded. Alterations were described
and possibly photographed. Females which did not litter within about one week after the expected end of gestation were necropsied and their uteri examined for implantation sites according to Salewski. All females with litters were necropsied after the 23rd day of lactation and the uteri were examined for implantation sites as above.

Statistics:

The statistical evaluations were carried out with the aid of the T-Test for independent samples and/or of the chi-Square-Test (Fisher's exact probability test). The data shown in the tables are calculated mean values and standard deviations.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

A slight nervousness of the whole group was reported at 150 mg/kg bw/d for day 13-15 and at 500 mg/kg bw/d for day 11-15 after treatment

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No compound related maternal death occurred in the 50 and 500 mg/kg dose groups. One pregnant female in the 150 mg/kg dose group died from unknown reasons on day 10 of gestation

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

In 500 mg/kg dose group a slight but statistically unsignificant increase of embryonic resorptions was assessed in the necropsy sub group but the value does not exceed the range of our historical control data.

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

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

behaviour (functional findings)

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

In the rearing group the only finding diverging from control group data was a very slight increased mortality of the pups of the 150 mg/kg dose group between day 1 and 4 of lactation, but the overall mortality between lactation days 1 to 23 was comparable to that of the concurrent control group. Because of this view point and because of a lack of dose dependence this result is to be regarded as random without biological relevance.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No skeletal abnormalities were observed in any dose group, except 1 fetus in the 50 mg/kg dose group, which showed various signs of bone development retardation as well as a cleft palate. Because of the single occurrence of these retardations and malformation in only one fetus of the lowest treatment group this is considered to be accidental.

Visceral malformations:

no effects observed

Description (incidence and severity):

The examination for soft tissue abnormalities revealed no indication of any drug-related deviation in any dose group

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity and maternal developmental toxicity

Effect level:

>= 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Table 1. Summary of the reproduction data. Young were examined for skeletal and soft tissue anomalies (sub group I) and for functional anomalies (subgroep II, rearing group). The females reveived daily doses of the test substance from day 7 to day 16 of gestation.

	CONTROL P.O.	50 MG/KG P.O.	150 MG/KG P.O.	500 MG/KG P.O.
Number of mated females Mortality during the experiment Number of surviving females Number of pregnant females (%) Mean starting weight of females with litter Mean weight gain in g Day of gest. 1-7 (before treatment) Day of gest. 7-17 (during treatment) Day of gest. 17-21 (after treatment Day of gest. 1-21 (total)	36 0 36 31 (86.1%) 213.8±9.2   24.7±4.7 42.2±6.2 48.6±9.9 115.5±13.7	36 1 35 31 (88.6 %) 215.1±7.5   24.6± 5.1 46.7±8.6 50.5±6.5 121.9±13.8	36 2 34 31 (91.2 %) 214.3±10.8   24.9±5.6 45.4±8.3 49.7±7.4 120.0±13.4	36 2 34 33 ( 97.1 %) 210.8±10.6   23.3± 4.3 42.5±10.2 44.1±21.6 109.8±28.0

Table 2. Summary of the reproduction data. Young were examined for skeletal and soft tissue anomalies (sub group I) and for functional anomalies (subgroep II, rearing group). The females reveived daily doses of the test substance from day 7 to day 16 of gestation.

Subgroup I: Examination for skeletal and soft tissue anomalies	CONTROL P.O.	50 MG/KG P.O.	150 MG/KG P.O.	500 MG/KG P.O.
Number of pregnant females Number of corpora lutea, total per pregnant female Number of implantations, total per pregnant female Total number of foetuses (Alizarin/Wilson) Live foetuses, total per pregnant female Dead foetuses, total per pregnant female Sex ratio m/f Number of resorptions, total per pregnant female Resorptions in % of implantations Developmental stage of resorption   Females with complete resorptions Mean litter weight in g Mean body weight of live foetuses	15 214 14.3 ±1.6 204 13.6±2.2 186 (96/90) 186 12.4 ± 2.5 0 0.0 75/111 18 1.2 8.8% 13x embryonic, 5x foetal   0 41.6 ± 8.7 3.4 ±0.2	15 225 15.0 ±2.0 218 14.5 ±1.7 201 (104/97) 201 13.4 ±1.5 0 0.0 101/100 17 1.1 7.8% 17x embryonic, 0x foetal 0 45.7 ± 5.1 3.4 ±0.2	15 227 15.1 ±1.6 214 14.3 ±2.0 200 (103/97) 200 13.3 ±2.1 0 0.0 102/98 14 0.9 4.5% 14xembryonic, 0x foetal 0 46.4 ± 6.5 3.5 ±0.2	15 222 14.8 ±1.4 218  14.5 ±1.6 187 (98/89) 187 12.5 ±3.5 0 0.0 95/92 31 2.1 14.2% 30x embryonic, 1x foetal 0 43.2 ±13.2 3.4 ±0.3

Table 3.Summary of the reproduction data. Young were examined for skeletal and soft tissue anomalies (sub group I) and for functional anomalies (subgroep II, rearing group). The females reveived daily doses of the test substance from day 7 to day 16 of gestation.

Subgroup II: Rearing group	CONTROL P.O.	50 MG/KG P.O.	150 MG/KG P.O.	500 MG/KG P.O.
Number of pregnant females Number of implantations, total per pregnant female Number of resorptions, total per pregnant female Resorptions in % of implantations Females with complete resorptions Mean gestation period in days Number of pups at lac. day 1, (birth), total per pregnant female Number of pups at lac. day 4, total per pregnant female Number of pups at lac. day 12, total per pregnant female Number of pups at lac. day 23, total per pregnant female Number of pups at lac. day 23 in % of implant, in % of pups born total Sex ratio m/f Mean litter weight in g At lact. Day 1 At lact. Day 4 At lact. Day 12 At lact. Day 23   Mean body weight of weight of pups in g: At lact. Day 1 At lact. Day 4 At lact. Day 12 At lact. Day 23	16 219 13.7± 2.1 16 1 7.3% 0 22.4 ±0.5 203 12.7 ± 1.9 2001 12.6 ± 1.7 191 11.9 ± 1.8 190 11.9 ±1.9 86.8 % 93.6 % 94/96   74.4±11.3 90.8 ±14.0 236.8 ±39.9 516.3 ±77.1     5.9 ±0.4 7.2 ±0.7 19.9 ±1.9 43.8 ±4.0	16 216 13.5±1.4 17 1.1 7.9% 0 22.4 ±0.5 199 12.4 ±1.9 193 12.1±1.8 184 11.5 ± 2.1 183 11.4 ±2.0 84.7% 92% 89/94   69.8± 12.0 86.7 ±16.6 227.4±40.5 509.0 ±75.4     5.6 ±0.4 7.2 ±0.7 19.9 ±2.0 45.0 ±4.7	16 207 12.9±2.7 24 1.5 11.6% 0 22.8 ±0.5 183 11.4± 2.9 171 10.7 ±3.1 167 10.4 ±3.0 166 10.4 ±3.1 80.2% 90.7% 91/75   67.8±16.1 79.8 ±21.7 209.7 ±55.3 461.2±120.1     6.0±0.5 7.6 ±1.2 20.4 ±2.6 45.5 ±5.6	18 217 12.1±2.8 12 0.7 5.5% 0 22.4 ±0.7 205 11.4 ±3.6 200 11.1 ±3.7 197 10.9 ±3.6 197 10.9 ±3.6 90.8% 96.1% 104/93   65.5±18.9 83.1 ±25.5 221.1±64.4 480.8 ±138.3     5.9 ±0.6 7.6 ±1.0 20.7± 2.8 45.0±6.4

Conclusions:

The NOAEL was set at 50 mg/kg for maternal effects based on slight nervousness and 500 mg/kg for foetal effects.

Executive summary:

The test substance was tested for embryotoxic and teratogenic action in accordance with OECD 414 and performed under GLP principles. In order to determine post-natal manifestations of prenatal administration of the test substance in albino rats, this investigations included concurrent rearing of offspring until weaning. Doses of 50, 150 and 500 mg/kg bw/day were administered daily by oral gavage from day 7 to day 16 of gestation. A control group received the vehicle SSV (Standard Solvent Vehicle: 4% carboxylmethylcellulose, 0.9% NaCl, 0.5% benzylalcohol, 0.4% Tween 80 in H2O dest).

The test substance was well tolerated in all dose groups. The only treatment related observation was a slight nervousness after treatment; in 500 mg/kg dose group from the 11th day of gestation and in the 150 mg/kg dose group from the 13th day of gestation. One animal in the 150 mg/kg dose group died from unknown reasons. Body weight development of the dams receiving treatment did not show deviations from the concurrent control group. There were no adverse effects noted on any of the measured reproductive or litter parameters for the 50, 150 and 500 dose groups, except a slightly increased mortality rate of the pups of the 150 mg/kg dose group between days of lactation 1 and 4. This effect is not dose-dependent and probably not of biological significance, because the overall mortality of the pups between days 1 and 23 of lactation is comparable to concurrent control group results and similar observations were not made in higher dose group. A slight increase of the number of embryonic resorptions was noted in the 500 mg/kg necropsy subgroup. This increase is not statistically significant, does not exceed the range of our historical data, and could not be confirmed by the resorption rate found in the comparable rearing group. It is considered as biologically insignificant. Macroscopic and soft tissue examination from treatment and control groups revealed one foetus oedematous with ectopia of the viscera in the control group and one foetus oedematous with various skeletal in the 50 mg/kg dose group. Skeletal examinations of all foetuses revealed no teratological findings in any dose group.

In conclusion, under the condition of the present study, the test substance when applied orally to pregnant rats up to a dose of 500 mg/kg/day is neither embryotoxic, teratogenic nor impairs the postnatal development of the offspring. The NOAEL was set at 50 mg/kg for maternal effects based on slight nervousness and 500 mg/kg for foetal effects.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

A guideline study (comparable to OECD 414) performed in compliance with GLP

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

Developmental toxicity test in rabbits, Hummler and Mc Kinney 1984

The test substance was tested for embryotoxic and teratogenic action in 20 mated (principle study) and 35 (supplementary study) female Swiss hare rabbits according to OECD TG 414 and GLP principles. In the principle study, doses of 30, 100 and 300 mg/kg bw/day were administered by oral gavage to pregnant animals from day 7 to 19 inclusive of gestation. A control group received the vehicle (SSV, 4% carboxymethylcellulose, 0.9% NaCl, 0.5% benzylalcohol, 0.4% Tween 80 in water) for the same treatment period. All females were sacrificed at day 30 of gestation. Foetuses were removed by ovariohysterectomy tested for viability (24 h) and examined for macroscopic, skeletal and soft tissue anomalies. Accurate interpretation of the findings of the principle study was increasingly complicated by the appearance of some malformations in the 300 mg/kg dose group which rendered inconclusive results. Therefore, a supplementary study was conducted with increased number of females using a dose of 200 mg/kg bw/day in an attempt to reproduce the same effects and to establish the relevance of the malformations that occurred in the 300 mg/kg group. The same treatment schedule was used as in the principle study and included a separate control group. Throughout the summary the principle and supplementary study will be referred as A and B.

Results showed a moderately reduced body weight development during the treatment period among females of the 200 and 300 mg/kg dose groups, but not a highly significant effect. There were no detectable effects on the measured reproductive parameters, on the course and outcome of pregnancy nor on the 24 hour survivability of the neonates in either study. In the principle study (A) beginning with a dose of 100 mg/kg, some morphological malformations appeared mainly as open eyes, spina bifida and/or short or missing tail. The incidence was low and compared favourably to the frequency of these malformations in observed historical control data of this rabbit strain. In spite of no effects seen in the preliminary study at a dose of 300 mg/kg, malformations did appear at this dose level in the principle study. The malformations were similar to those observed in the 100 mg/kg group but the incidence was somewhat higher than recognized in historical controls thus, complicating an accurate interpretation of the results. In the supplementary study (B) using a dose of 200 mg/kg, it was assumed that a pattern of the same types of malformations would appear if the effects seen in the 300 mg/kg group were relevant. However, the results from this study show that no pattern or associated malformation was established between the two doses and only one malformation was present and localized in a foetal resorption. Furthermore, in contrast to the single malformation from the control group of the principle study (A), the malformations seen in the control group of the study supplementary study (B) appeared more frequently and were analogous in both type and incidence to the malformations seen in the 300 mg/kg dose group. Therefore, in view of all substantiating evidence with particular emphasis on the high incidence of malformations seen in the controls of the supplementary study, all malformations seen in the principle study (A) of the 300 mg/kg group are considered to be arbitrary findings and not test substance-related. Examination of the foetuses for skeletal and soft tissue anomalies did not reveal any effects considered to be test substance-related from any dose group.

In conclusion, based on decreased body weight, the NOAEL for maternal toxicity was set at 100 mg/kg bw/day. Based on no effects on foetal development the NOAEL for developmental toxicity was set at ≥ 300 mg/kg bw/day.

Developmental toxicity test in rats, Eckhardt 1983

The test substance was tested for embryotoxic and teratogenic action in accordance with OECD 414 and performed under GLP principles. In order to determine post-natal manifestations of prenatal administration of the test substance in albino rats, this investigations included concurrent rearing of offspring until weaning. Doses of 50, 150 and 500 mg/kg bw/day were administered daily by oral gavage from day 7 to day 16 of gestation. A control group received the vehicle SSV (Standard Solvent Vehicle: 4% carboxylmethylcellulose, 0.9% NaCl, 0.5% benzylalcohol, 0.4% Tween 80 in H2O dest).

The test substance was well tolerated in all dose groups. The only treatment related observation was a slight nervousness after treatment; in 500 mg/kg dose group from the 11th day of gestation and in the 150 mg/kg dose group from the 13th day of gestation. One animal in the 150 mg/kg dose group died from unknown reasons. Body weight development of the dams receiving treatment did not show deviations from the concurrent control group. There were no adverse effects noted on any of the measured reproductive or litter parameters for the 50, 150 and 500 dose groups, except a slightly increased mortality rate of the pups of the 150 mg/kg dose group between days of lactation 1 and 4. This effect is not dose-dependent and probably not of biological significance, because the overall mortality of the pups between days 1 and 23 of lactation is comparable to concurrent control group results and similar observations were not made in higher dose group. A slight increase of the number of embryonic resorptions was noted in the 500 mg/kg necropsy subgroup. This increase is not statistically significant, does not exceed the range of our historical data, and could not be confirmed by the resorption rate found in the comparable rearing group. It is considered as biologically insignificant. Macroscopic and soft tissue examination from treatment and control groups revealed one foetus oedematous with ectopia of the viscera in the control group and one foetus oedematous with various skeletal in the 50 mg/kg dose group. Skeletal examinations of all foetuses revealed no teratological findings in any dose group.

In conclusion, under the condition of the present study, the test substance when applied orally to pregnant rats up to a dose of 500 mg/kg/day is neither embryotoxic, teratogenic nor impairs the postnatal development of the offspring. The NOAEL was set at 50 mg/kg for maternal effects based on slight nervousness and 500 mg/kg for foetal effects.

Justification for classification or non-classification

Based on the available information, classification is not required for the test substance in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.