2-methyl-1-(4-methylthiophenyl)-2-morpholinopropan-1-one

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The study provided was conducted to recognised testing guidelines and with GLP certification.

Link to relevant study records

Reference

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Sprague Dawley SD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Italy s.r.l., San Pietro al Natisone (UD), Italy
- Age at study initiation: males: approx. 7 - 8 weeks; females: approximately 10 - 11 weeks
- Weight at study initiation: females: 217.1 - 264.0 g
- Housing: 4 of one sex to a cage
- Diet (e.g. ad libitum): laboratory rodent diet (Altromin MT pelleted diet, Lage, Germany) was offered ad libitum throughout the study
- Water (e.g. ad libitum): Drinking water was supplied ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12 hours/12 hours

Route of administration:

oral: gavage

Vehicle:

other: Aqueous solution of 0.5 % carboxymethylcellulose (CMC).

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The required amount of test substance were suspended in the vehicle. The formulation will be prepared daily (concentrations of 4.0, 8.0 and 12.0 mg/ml). Concentrations will be calculated and expressed in terms of test item as received. All test item solutions were protected from light throughout gavage.

DOSE VOLUME: The test item was administered at a dose volume of 10 ml/kg body weight. Control animals received the vehicle alone at the same dose volume. Dose volumes were calculated according to individual body weight on the first day of freatment and adjusted according to individual body weight at weekly intervals thereafter.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: monogamous (1 male to 1 female)
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility. One treated male was utilised to mate a maximum of three females.
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy. These data also provided information about the pre-coital interval (i.e. the number of nights paired prior to the detection of mating).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the formulations prepared during the first and the last week of treatment were analysed to check the concentration. The test item was found to be stable in the vehicle for 4 hours at room temperature. Chemical analyses were carried out by the Analytical Chemistry Department at RTC. The results of analyses were within the limits of acceptance of +/- 10% of nominal concentration.

Duration of treatment / exposure:

males: for 10 consecutive weeks prior to mating and thereafter through the day prior to sacrifice.
females: for 2 consecutive weeks prior to mating and thereafter: half of the females per group until post-coitum day 19 and the other half of the females per group which were allowed to give birth until the day before necropsy (Day 21 post-partum).

Frequency of treatment:

once daily, 7 days a week

Details on study schedule:

Culled pups were killed on Day 4 post-partum, weaned pups on Day 21 post-partum.

Remarks:

Doses / Concentrations:
0, 40, 80, 120 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

Each group comprised 48 females and 24 males, with the exception of the female control group which comprised 24 females.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on information from the preliminary study (RTC Study No.: 9318EXT).

Positive control:

no

Parental animals: Observations and examinations:

MORTALITY: Yes
- Time schedule: twice daily (early in each working day and again in the afternoon). At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.

PRE- AND POST-DOSE OBSERVATIONS: Yes
- Time schedule: daily, prior to dosing, immediately after dosing and within 1 hour of dosing
- Observations included: any changes in gait and posture, reactivity to handling, stereotypes or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern)

DETAILED CLINICAL OBSERVATIONS: Yes
-Time schedule: once a week
- Observations included: any changes in gait and posture, reactivity to handling, stereotypes or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). In addition, abnormal changes of the skin/fur (abnormal coloration, presence of scabs, hairless, erythema, oedema, necrosis) with relative localisation and presence of palpable masses with relative localisation were recorded.

BODY WEIGHT: Yes
- Time schedule: Males = on the day of allocation to treatment group, on the day that treatment commenced, weekly thereafter and just prior to necropsy. Females = on the day of allocation to treatment group, on the day that treatment commenced, weekly thereafter until pairing. Body weight during the mating period were recorded, but not presented.
The females were weighed on days 0, 3, 6, 9, 12, 15, 18 and 20 post-coitum and on days 1, 7, 14 and 21 post-partum.

FOOD CONSUMPTION:
- Time schedule: weekly from allocation to pairing; females: Individual food consumption was also measured on days 0, 3, 6, 9, 12, 15 and 20 post-coitum and days 1,7, 14 and 21 post-partum.

ORGAN WEIGHTS: From all males completing the scheduled test period, the testes and epididymides were weighed. The ratios of organ weight to body weight were calculated for each animal.

Oestrous cyclicity (parental animals):

Vaginal smears were taken daily in the morning, starting from 2 weeks before pairing up to the end of the mating period, until a positive identification of mating was made. The vaginal smear data were examined to determine anomalies of the oestrous cycle.

Sperm parameters (parental animals):

Examination confined to testes and epididymides weights

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: Litter size, sex ratios, number of foetuses affected with structural deviations and the corresponding litter percentage, foetal structural deviations (Malformations, Abnomalities, Variations), stillbirths, total litter loss, litter weight, pre-weaning development
Dead pups were grossly examined for external and internal abnormalities; sex confirmation by gonadal inspection

Postmortem examinations (parental animals):

All females selected to give birth were examined externally and internally, for the number of visible implantation sites and number of corpora lutea (if detectable).
Procedure for females sacrificed on gestation Day 20 - the ovaries and uteri will be examined to determine: a) number of corpora lutea; b) weight of intact gravid uterus; c) number and distribution of live young; d) number and distribution of intra-uterine deaths; e) individual foetal weight and sex; f) external foetal abnormalities and g) gross evaluation of placentae.
A selection of tissues were fixed and preserved. Histopathological examination was not performed.

Postmortem examinations (offspring):

All foetuses will be examined externally as well as for visceral and skeletal abnormalities.

Statistics:

For continuous variables the significance of the differences amongst group means will be assessed by Dunnett's test or a modified t test, depending on the homogeneity of data.
Statistical analysis of non-continuous variables will be carried out by means of the Kruskal-Wallis test and intergroup differences between the control and treated groups assessed by a non-parametric version of the Williams test.
A modified chi-squared test was used for fertility index parameter. The criterion for statistical significance was p<0.05.

Reproductive indices:

Copulatory Index (%), Fertility Index (%), Copulatory Interval, Oestrus cycles, Pre-implantation loss, Post-implantation loss, total implantation loss

Offspring viability indices:

Pup loss, cumulative pup loss, lactation index

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Hairloss and swollen abdomen were noted in the high dose females during the gestation period. Staining on the body surface was observed in mid- and high dose females during gestation and the post partum phase.
One high dose male and one mid-dose female were sacrificed for humane reasons on Day 72 of the study and on Day 0 post-partum, respectively. Difficulty in parturition was noted for the female and reduced activity and pallor were noted in the male. No clinical signs of particular relevance were noted in males, throughout the treatment period, or in females before pairing. Hairloss and swollen abdomen were noted in the high dose females during the gestation period. Staining on the body surface was observed in mid- and high dose females during gestation and the post partum phase. Body weight, body weight gain and food consumption in male and female animals was comparable to control.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

Pre-coital interval and copulatory index were comparable between groups and sexes. Fertility was decreased in all treated females and significantly decreased in the high dose group. An increase in irregular cycle (not significant, out of historical range) was noted in all treated females. The number of implantation sites decreased in a dose dependent manner, with the effect still being within the historical range and considered of no statistical significance. Gestation periods were similar in the treated groups compared to controls. All dams gave birth between Day 21 and 22 post-coitum.
A total of 18 females which were allowed to give birth proved not to be pregnant at sacrifice (three low dose, three mid dose and twelve high dose group. The number of females with live foetuses on day 20 post-coitum was 12 in the control, 21 in the low dose, 21 in the mid dose and 13 in the high dose group.
There is no indication from this study that male fertility was affected. Testes and epididymides weights were unaffected and repeat-dose studies also did not indicate adverse effects on male reproductive organs.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Dose descriptor:

LOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: reduced fertility, increased pre-birth loss, reduced number of implantations

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: slight reduction in the number of implantations and increase in pre-birth loss

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Stillbirths or total litter loss was noted at the day of parturition or the day after parturition in all high dose females which gave birth. A significant increase in pup loss on day 1 post-partum, cumulative loss on days 4 and 14 post-partum was observed in the mid-dose group. Litter weight was significantly decreased in the mid-dose group on days 4 and 14 post-partum. In addition, decreases in the number of viable males and consequently in the percentage of males were noted for the mid- group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Slight retardation in the development of pups (Pinna detachment, hair growth and upper incisor emption) was noted in the mid-dose group (without statistical significance).

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

In decedent pups no milk in the stomach was observed during the necropsy. The incidence of this finding increased with the treatment. In addition, head with domed shape, hydrocephaly, dilatation of the cerebral ventricle and small cerebrum were the findings observed in the decedent pups of the mid-dose group. In the high dose group, these pups showed forelimbs or hindlimbs short and malrotated, head with domed shape, cleft palate, micrognathia, kyphosis, tail short or bent, short body and anasarca. Tail bent was also noted in the mid-dose group. In some cases, autolysis did not allow necropsy to be performed.
Lateral ventricles or third ventricle of the brain dilated and abnormal size of the urinary bladder in weaned pups of the low- and mid-dose groups. In individual cases, innominate artery was not evident.

Histopathological findings:

not examined

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

80 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: sign. increase in pup loss, sign. reduced litter size and litter weight

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

40 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: reduction in total litter size

Reproductive effects observed:

not specified

dose group	total litter size at birth	litter size live (day 1 p.p.)	litter size live (day 21 p.p.)	litter weight (day 1 p.p.)	liter weight (day 4 p.p.)	% males at birth	% males (day 4 p.p.
control	15.0	13.1	7.8	83.9	116.4	44.22	45.47
40 mg/kg bw	13.9	12.7	7.7	84.2	120.1	44.14	43.04
80 mg/kg bw	12.3	6.6*	0	48.2	61.5*	52.17	46.31
120 mg/kg bw	11.9	-	-	-	-	54.45	-

* p < 0.05 adjustment of litter size on day 4 p.p. to n = 8 (4 males, 4 females)

dose group	fertility index (M)	fertility index (F)	irregular cycles	implantation	pre-birth loss %	females pregnant/total
control	83.3	91.7	3/24	16.33	8.03	12/12
40 mg/kg bw	100	87.5	12/48	15.4	8.64	21.24
80 mg/kg bw	95.8	87.5	11/48	15.05	17.23	21/24
120 mg/kg bw	82.6	64.6*	14.48	13.91	14.63	13/25
hist. control	83.3 -91.7	83.3 -91.7	2 -5/24	12.7 -16.7	3.5 -6.8

* = significant at p < 0.05

Conclusions:

The reproduction toxicity NOAEL of the test material in rats was determined to be 40 mg/kg bw/day based upon number of implatations, litter size, and number of still births under the conditions of the test.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

40 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

1

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a GLP conform combined 1-Generation / developmental toxicity study according to OECD guideline 414 and 415 (Research Toxicology Centre S. p. A, 2004) effects of the test substance on male and female reproductive performance and effects of prenatal exposure on the pregnant test animal and on the developing organism were investigated. The test substance was administered daily by gavage to groups of male rats and female rats at concentrations of 0 (control), 40, 80 and 120 mg/kg/day, based on a dose finding study. The males were dosed for 10 consecutive weeks before pairing and during pairing until termination. The females were dosed for 2 consecutive weeks before pairing and during pairing until Day 19 post-coitum (developmental part) oruntil weaning (fertility part), respectively.

Systemic toxicity was evident by decreased body weight and body weight gain in mid- and high dose animals; food consumption was also reduced. Clinical signs were limited to the high dose females and consisted of stained body surface, alopecia and swollen abdomen. Macroscopic observation of the sacrificed animals did not reveal treatment related findings.

Fertility, in general, was decreased in all treated females and significantly decreased in the high dose group. The number of implantations was possibly decreased in the high dose group and an increase in pre-birth loss was noted for mid- and high dose animals. In addition, an increase in irregular oestrus cycle was noted in all treated females. The NOAEL for maternal toxicity and fertility is considered to be 40 mg/kg bw/day.

Total litter size and implantation size were reduced in all three groups. A total litter loss at the day of parturition or day one p.p. was recorded for all high dose females. Increased litter loss was also observed for the mid-dose females; by day 21 p.p. all pups deceased. Weights of the remaining mid-dose litters we significantly reduced and a decrease in the number of viable males and consequently in the percentage of males wasnoted for this treatment group.Additionally, slight retardation in the development of pups was noted in the mid-dose group.Several malformations e.g. domed shape, cleft palate, abnormal brain development weremainly noted in the high dose group and to a lesser extent in the mid dose group. But malformations did also occur in individual litters of the low-dose group.In decedent pups no milk in the stomach was observed during the necropsy. The incidence of this finding increased with the treatment.

Several findings concerning the F1 generation underpin the low likelihood of a lactation effect but support a perinatal toxicity effect due to parental/maternal toxicity, i.e.cumulative offspring mortality and litter loss on days 0, 1, 2 p.p. in the mid-dose group and reduced food intake in dams of the mid-dose group during the lactation period. Furthermore, observations on the pups during lactation („neglected pups“) and necropsy („apparently no food intake”) indicate nursing deficits of the female rats. Therefore, damage to the offspring during lactation cannot clearly be derived from the data.

Short description of key information:
In a GLP conform combined 1-Generation / developmental toxicity study according to OECD guideline 414 and 415 effects of the test substance on male and female reproductive performance and effects of prenatal exposure on the pregnant test animal and on the developing organism were investigated. Fertility, in general, was decreased in all treated females and significantly decreased in the high dose group. The number of implantations was possibly decreased in the high dose group and an increase in pre-birth loss was noted for mid- and high dose animals. In addition, an increase in irregular oestrus cycle was noted in all treated females. The NOAEL for maternal toxicity and fertility is considered to be 40 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

The study provided was conducted to recognised testing guidelines and with GLP certification.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

other: OECD guideline 415 (One-Generation Reproduction Toxicity Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Sprague Dawley SD

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Italy s.r.l., San Pietro al Natisone (UD), Italy
- Age at study initiation: males: approx. 7 - 8 weeks; females: approximately 10 - 11 weeks
- Weight at study initiation: females: 217.1 - 264.0 g
- Housing: 4 of one sex to a cage
- Diet (e.g. ad libitum): laboratory rodent diet (Altromin MT pelleted diet, Lage, Germany) was offered ad libitum throughout the study
- Water (e.g. ad libitum): Drinking water was supplied ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12 hours/12 hours

Route of administration:

oral: gavage

Vehicle:

other: Aqueous solution of 0.5 % carboxymethylcellulose (CMC).

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The required amount of test substance were suspended in the vehicle. The formulation will be prepared daily (concentrations of 4.0, 8.0 and 12.0 mg/ml). Concentrations will be calculated and expressed in terms of test item as received. All test item solutions were protected from light throughout gavage.

DOSE VOLUME: The test item was administered at a dose volume of 10 ml/kg body weight. Control animals received the vehicle alone at the same dose volume. Dose volumes were calculated according to individual body weight on the first day of treatment and adjusted according to individual body weight at weekly intervals thereafter.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the formulations prepared during the first and the last week of treatment were analysed to check the concentration. The test item was found to be stable in the vehicle for 4 hours at room temperature. Chemical analyses were carried out by the Analytical Chemistry Department at RTC. The results of analyses were within the limits of acceptance of +/- 10% of nominal concentration.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: monogamous (1 male to 1 female)
- Length of cohabitation:
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility. One treated male was utilised to mate a maximum of three females.
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy. These data also provided information about the pre-coital interval (i.e. the number of nights paired prior to the detection of mating).

Duration of treatment / exposure:

males: for 10 consecutive weeks prior to mating and thereafter through the day prior to sacrifice.
females: for 2 consecutive weeks prior to mating and thereafter: half of the females per group until post-coitum day 19 and the other half of the females per group which were allowed to give birth until the day before necropsy (Day 21 post-partum).

Frequency of treatment:

once a day, 7 days a week

Duration of test:

Males were killed after 15 weeks of treatment.
Approximately half of the females per group were sacrificed on Day 20 post-coitum.
Approximately half of the females per group were allowed to give birth.

Remarks:

Doses / Concentrations:
0, 40, 80, 120 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

Each group comprised 48 females and 24 males, with the exception of the female control group which comprised 24 females.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on information from the preliminary study (RTC Study No.: 9318EXT).

Maternal examinations:

MORTALITY: Yes
- Time schedule: twice daily (early in each working day and again in the afternoon). At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.

PRE- AND POST-DOSE OBSERVATIONS: Yes
- Time schedule: daily, prior to dosing, immediately after dosing and within 1 hour of dosing
- Observations included: any changes in gait and posture, reactivity to handling, stereotypes or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern)

DETAILED CLINICAL OBSERVATIONS: Yes
-Time schedule: once a week
- Observations included: any changes in gait and posture, reactivity to handling, stereotypes or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). In addition, abnormal changes of the skin/fur (abnormal coloration, presence of scabs, hairless, erythema, oedema, necrosis) with relative localisation and presence of palpable masses with relative localisation were recorded.

BODY WEIGHT: Yes
- Time schedule: Males = on the day of allocation to treatment group, on the day that treatment commenced, weekly thereafter and just prior to necropsy. Females = on the day of allocation to treatment group, on the day that treatment commenced, weekly thereafter until pairing. Body weight during the mating period were recorded, but not presented.
The females were weighed on days 0, 3, 6, 9, 12, 15, 18 and 20 post-coitum and on days 1, 7, 14 and 21 post-partum.

FOOD CONSUMPTION:
- Time schedule: weekly from allocation to pairing; females: Individual food consumption was also measured on days 0, 3, 6, 9, 12, 15 and 20 post-coitum and days 1,7, 14 and 21 post-partum.

ORGAN WEIGHTS: From all males completing the scheduled test period, the organs were dissected free of fat and weighed. The ratios of organ weight to body weight were calculated for each animal.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day: on day 20 post-coitum.
- Organs examined: detailed post-mortem examination was conducted (including examination of the external surface and orifices)

Ovaries and uterine content:

The ovaries and uterine content was examined after termination
Examinations included:
- Gravid uterus weight, number of corpora lutea, number and distribution of live young, number and distribution of intra-uterine deaths, individual foetal weight and sex
- External foetal abnormalities: Yes
- Gross evaluation of placentae: Yes

Fetal examinations:

BODY WEIGHT:
- Time schedule: on day 1 post-partum

EXTERNAL EXAMINATIONS: Yes, for dead or abnormal young
- Time schedule: daily
- Observations included: pinna unfolding, hair growth, incisor eruption (upper), startle response to sound, eye opening (complete separation of eyelids), testis descent (testes palpable in the scrotum), air righting reflex, pupil reflex.

STRUCTURAL DEVIATIONS: Observations included: malformations, abnomalities, variations

NECROPSY: all pups found dead were necropsied with the exception of those excessively cannibalized or autolysed.

Statistics:

For continuous variables the significance of the differences amongst group means will be assessed by Dunnett's test or a modified t test, depending on the homogeneity of data.
Statistical analysis of non-continuous variables will be carried out by means of the Kruskal-Wallis test and intergroup differences between the control and treated groups assessed by a non-parametric version of the Williams test.

Historical control data:

yes

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Before pairing, no clinical signs of toxicological significance were noted. During the gestation period, staining on the body surface was observed in some treated females. This observation continued during the post-partum phase in the mid- and high-dose groups. In addition, hair loss, and swollen abdomen were noted in high dose females.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

During the gestation period, statistically significant decreases in body weight were observed in mid- and high dose females from day 15 to 20 and from day 3 to 20 post coitum, respectively. Mean group values for the mid- and high dose terminal body weight were 7.4 and 8.5% lower than the control mean value, respectively (tab. 1). Food intake was unaffected by treatment in both sexes before pairing and in females during post-coitum period.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

A total of 13 females proved not to be pregnant at sacrifice. The number of females with fetuses on day 20 post-coitum was 10 in the control, 21 in the low dose, 21 in the mid dose and 18 in the high dose group (tab 2). The number of corpora lutea was decreased in all treatment groups. Pre-, post and total implantation loss in all treatment groups was comparable to the control. A significant lower uterus weight was observed in the mid-dose group; the absolute uterus weight gain was decreased in mid- and high-dose females (tab. 2).

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Pre-, post and total implantation loss in all treatment groups was comparable to the control.

Dose descriptor:

LOAEL

Effect level:

40 mg/kg bw/day (nominal)

Basis for effect level:

other: developmental toxicity

Dose descriptor:

LOAEL

Effect level:

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

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

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

Basis for effect level:

other: maternal toxicity

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Litter weight was statistically significantly decreased in the mid-dose and high dose group. Mean foetal weight was statistically significantly decreased in the high dose group when compared to controls (tab 3).

Changes in sex ratio:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant decreases in the number of viable males and consequently in the percentage of males and litter weight were noted in the mid- and high dose groups (tab 3).

External malformations:

effects observed, treatment-related

Description (incidence and severity):

Micrognathia, cleft palate, anasarca, tail bent, short or swollen, short body, kyphosis, limbs (forelimbs and/or hindlimbs) malrotated, short or flexure and head with domed shape were observed in the high dose foetuses. In addition, cleft palate was also noted in 5 low dose foetuses and in 1 mid-dose foetus out of one litter each. Anasarca was observed in the low dose group as well as head with domed shape and hindlimbs malrotated in the mid-dose group. A total of 13 small foetuses were present; 2 each in the low and mid-dose groups and 9 in the high dose group (tab. 4).

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Retardation or no ossification of the sternal elements with cases of asymmetrical ossification were observed in all treated groups. In addition, an increased incidence in general incomplete ossification of the head bones was noted in the high dose group compared to controls. Alteration of ossification in the vertebral column (thoracic, cervical lumbar and sacral vertebrae) with cases of scoliosis were observed in the high dose group.
Rudimentary 14th rib was noted in all treated groups. Displaced ribs were also observed in the high dose group. Dose-related metacarpal(s) incomplete ossification or unossified were observed in all treated groups (tab 6). Metatarsal(s) incomplete ossification or unossified and incomplete ossification of the pubis bone were noted in the high dose group.
A variety of abnormalities and/or variations were recorded especially in mid and high-dose groups, e.g. retarded / incomplete ossification at various sites of the skeleton. These abnormalities/ variations were not addressed individually in this summarized data compilation since they are of minor relevance for classification as compared to the severe teratogenic effects.

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

A dome-shape observed during external examination was associated with malformations of the brain. In particular, an increased incidence of enlarged lateral, third and fourth vehicles were observed in mid- and high-dose groups. High-dose foetuses also showed cases of anencephaly, anophthalmia and microphthalmia. Cleft palate and abnormal shape of the fore- and hindlimbs as well as short digits were detected in high-dose foetuses.
An increased incidence in pelvic dilatation of the kidneys with ureters enlarged and/or kinked were noted in all treatment groups. In addition, testes and kidneys displaced were also noted. Criptorchism, kyphosis and short body were noted in the high dose, as well as one case of malformation on the septum wall of the heart and on the intestine and an increased incidence of the agenesis of the pituitary gland (tab 5).

Dose descriptor:

NOEL

Effect level:

0 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

visceral malformations

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: skull

skeletal: forelimb

skeletal: vertebra

Developmental effects observed:

not specified

Table1 Body weight females, post-coitum period

	days post-coitum period
group	0	3	6	9	12	15	18	20
Control	242,42	260,88	272,24	284,5	299,46	321,3	368,4	404,05
40 mg/kg	237,25	253,74	266,31	279,01	293,05	311,35	254,78	387,37
80 mg/kg	241,96	258,9	267,06	278,03	291,35	308,84*	343,12**	375,78
120 mg/kg	232,03	248,99*	260,41*	269,16**	282,04**	299,78**	339,10**	371,33**

* = p< 0.05 ** = p< 0.01

Table 2 Pathology data females(females sacrificed on day 20 p.c.)

	control	40 mg/kg bw	80 mg/kg bw	120 mg/kg bw	Historical control data
Corpora lutea	17.6	15.9*	14.8*	15.2*	13.3-17.7
Implantations	16.5	15.1*	14.2*	14.7*	12.7-16.7
Pre-implantation loss %	5.9	5.6	4.0	3.2	3.5-6.8
Post-implantation loss %	3.8	4.6	10.1	6.2	2.6-6.3
Pre-birth loss %	8.03	8.64	17.23	14.63	3.5-6.8
Total implantation loss %	9.6	10.1	13.9	9.2	----
Gravid uterus weight [g]	94	83	77*	82	66.2-88.9
Absolute gain (uterus: BW ratio)	66	64	54*	55*	44.5-72.3
Females pregnant / total	10/12	21/24	21/24	18/24	-----

Table 3 Litter data – sex ratio, litter weight, fetal weight

Dose group	Total viable young / litter	Viable males %	Litter weight (g)	Mean fetal weight (g)
Control	15.9	55.3	62.3	3.9
40 mg/kg	14.3*	50.5	55.0	3.8
80 mg/kg	12.8*	40.8*	48.4*	3.8
120 mg/kg	13.8*	45.2*	48.5*	3.5*
Historical control data	12.0 – 15.6	48.0 – 54.2	42.1 – 59.3	3.5 – 3.8

* = p< 0.05

Table 4 Fetal malformations (1) (external examination, group incidence)

Organ	Malformation	Control	40 mg/kg	80 mg/kg	120mg/kg	Historical control data (%) #
	No. of fetuses (litters) examined	159 (10)	301 (21)	269 (21)	249 (18)
Forelimbs	Malrotated	0	0	0	27 (2)	n.f.
	Short	0	0	0	59 (5)	n.f.
	Flexure	0	0	0	8 (1)	n.f.
Head	Domed	0	0	7 (1)	47 (5)	n.f.
	Micrognathia	0	0	0	14 (1)	n.f.
Hindlimbs	Malrotated	0	0	1	47 (8)	n.f.
	Short	0	0	0	21 (4)	n.f.
Palate	Cleft palate	0	5 (1)	1 (1)	28 (5)	n.f.
Tail	Bent	0	0	0	46 (7)
	Short	0	0	0	29 (3)	n.f.
Whole foetus	Anasarca	0	7(1)	0	36 (4)	n.f.
	Short body	0	0	0	53 (4)	n.f.
	Kyphosis	0	0	0	18 (4)	n.f.

# Historical control data were generated from either the test laboratory or the breeder company n.f. = observation not found in the historical data set

Table 5 Malformations (2) (fixed foetuses)

Organ	Malformation	Control	40 mg/kg	80 mg/kg	120mg/kg	Historical control data (%) #
	No. of fetuses (litters) examined	77 (10)	145 (21)	128 (21)	121 (18)
Brain	Agenesis of the pituitary	0	0	1 (1)	27 (6)	n.f.
	Lateral ventricle enlarged, extreme	0	0	14 (5)	90 (16)	n.f.
	Third ventricle enlarged, extreme	0	0	2 (1)	52 (14)	n.f.
	Fourth ventricle enlarged, extreme	0	0	8 (3)	48 (14)	n.f.
	Anencephaly	0	0	0	10 (3)	n.f.
Eye	Anophthalmia	0	0	0	1 (1)	n.f.
Palate	Cleft palate	0	2 (1)	0	19 (5)	1 (1) = 0.1%
Heart	Interventricular septum wall incomplete	0	0	0	1(1)	n.f.
Abdomen	Diaphragmatic haernia	0	0	0	1	0.04 (0.26)
Limbs	Fore- and hindlimb abnormal shape	0	0	0	26 (6)	n.f.
Kidney	Pelvic dilatation extreme	0	0	0	7 (5)	n.f.
Ureter	Enlarged extreme	0	0	1 (1)	12 (7)	n.f.
	Kinked, extreme	0	0	0	4 (2)	0.37 (2.38)
Testis	Cryptorchism	0	0	0	20 (7)	n.f.
Forelimb	Short digit	0	0	0	8 (2)	n.f.
Intestine	Protrusion intestine from abdominal cavity	0	0	0	1 (1)	n.f.
Whole Foetus	Kyphosis	0	0	0	20 (6)	n.f.
	Short body	0	0	0	16 (3)	n.f.

# Historical control data were generated from either the test Lab or the Breeder company

n.f. = observation not found in the historical data set

Table 6 Malformations (3) (skeletal findings, group incidence)

Organ	Malformation	Control	40 mg/kg	80 mg/kg	120mg/kg	Historical control data (%) #
	No. of fetuses (litters) examined	82 (10)	156 (21)	141 (21)	128 (18)
Cervical vertebrae	Arches fused	0	0	0	1 (1)	0.04 (0.27)
Thoracic vertebrae	Scogliosis	0	0	0	22 (5)	n.f.
Rib	Displaced	0	0	0	21(5)	n.f.

# Historical control data were generated from either the test Lab or the Breeder company

n.f. = observation not found in the historical data set

Conclusions:

The developmental toxicity NOEL of the test material in rats was determined to be 0 mg/kg bw/day based upon statistically siognificant visceral and skeleton malformations in offspring at all test doses.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

40 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

1

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

In a GLP conform combined 1-Generation / developmental toxicity study according to OECD guideline 414 and 415 (Research Toxicology Centre S. p. A, 2004) effects of the test substance on male and female reproductive performance and effects of prenatal exposure on the pregnant test animal and on the developing organism were investigated. The test substance was administered daily by gavage to groups of male rats and female rats at concentrations of 0 (control), 40, 80 and 120 mg/kg/day, based on a dose finding study. The males were dosed for 10 consecutive weeks before pairing and during pairing until termination. The females were dosed for 2 consecutive weeks before pairing and during pairing until Day 19 post-coitum (developmental part) oruntil weaning (fertility part), respectively.

Systemic toxicity was evident by decreased body weight and body weight gain in mid- and high dose animals; food consumption was also reduced. Clinical signs were limited to the high dose females and consisted of stained body surface, alopecia and swollen abdomen. Macroscopic observation of the sacrificed animals did not reveal treatment related findings.

In females which were sacrificed by day 20 post-coitum,numbers of corpora lutea in treated animals are apparently differing (lower) from the numbers of the control group and display a statistical significance. By comparison with historical control data, study control values were located on the upper end of the normal range and the numbers for all treated groups are within the historical control range. Therefore, this finding is not considered to be of toxicological significance.

A significant lower gravid uterus weight was observed in the mid-dose group; the absolute uterus weight gain was decreased in mid- and high-dose females but was still within the historical data range.

Fetal examination revealed a decrease in the number of viable young per litter for all treatment groups. Litter weight was significantly lower in the mid- and high dose group; mean fetal weight was decreased in high dose offspring. In addition, the number of viable males of the mid- and high dose group was significantly decreased. Various external and visceral malformations were evident in the mid and high dose group and in individual litters of the low dose group. Skeletal findings were limited to the high dose group. A NOAEL for developmental toxicity is therefore not derived. The LOAEL is considered to be 40 mg/kg bw/day.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008:

The available experimental test data is reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is classified for developmental toxicity and fertility (Repr. 1B, H360Df) under Regulation (EC) No. 1272/2008.