3-methoxy-N,N-dimethylpropionamide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In the reproductive/developmental toxicity screening test with Wistar rats, conducted according to OECD guideline 421 and under GLP, no adverse changes on fertility were noted at the highest tested dose of 1000 mg/kg bw/day, therefore this dose level is considered to be a NOAEL for reproductive toxicity.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 January 2010 - 16 March 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study predates the most recent version of the guideline; thus, no evaluation of thyroid hormone levels were conducted.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

27 July 1995

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

dd 20 December 2008

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (15-25°C)

- Solubility and stability of the test substance in the solvent/vehicle: stable at 5±3°C pending use within 4 days (96 hours), or at room temperature pending use within 24 hours

Species:

rat

Strain:

Wistar

Remarks:

CRL:(WI) BR

Details on species / strain selection:

The rat is a suitable species for reproduction studies and the test guideline is designed to use the rat. Wistar rat was selected due to experience with this strain of rat in reproduction toxicity studies and known fertility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 Sulzfeld
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10-11 weeks old (males) and 11-12 weeks old (females)
- Weight at study initiation: (P) Males: 344-401 g; Females: 237-266 g
- Fasting period before study: no
- Housing: group-housed up to 5 animals/sex/cage in type II and III polypropylene/polycarbonate cages, with the exception of the mating and gestation/delivery period, when they were paired or individually housed, respectively. Deep wood sawdus was provided for bedding and nesting.
- Diet: ssniff® SM RJM-Z+H "Autoclavable complete feed for rats and mice-breeding and maintenance" produced by ssniff Spezialdiaten GmbH, D-59494, Soest, Germany, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23.3
- Humidity (%): 24-50
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 28 January 2010 To: 16 March 2010

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations were prepared at the appropriate frequency and stored refrigerated at 5±3°C pending use within 4 days (96 hours), or at room temperature pending use within 24 hours, according to stability assessment results.

VEHICLE

- Concentration in vehicle: 2.5, 15 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1:1, mating of siblings avoided
- Length of cohabitation: up to 7 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- No further mating performed after 7 days of unsuccessful pairing
- After successful mating each pregnant female was caged individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of formulations (concentration and homogeneity) was performed in the analytical laboratory of the test facility. Top, middle and bottom samples were taken in duplicate for analysis of concentration and homogeneity from each of the test-item dosing solutions on 3 occasions, during the first and last weeks of treatment for both males and females. Similarly, one sample was taken in duplicate on each occasion from the control solution, for concentration measurements. The analyses were performed by HPLC.

Duration of treatment / exposure:

Males: 28 days (14 days pre-mating and 14 days mating/post-mating)
Mated females: 14 days pre-mating, up to 7 days mating, throughout gestation and at least 4 days post-partum prior to necropsy.
Females showing no evidence of mating: 14 days pre-mating, up to 7 days mating, and 25-28 days after the last day of mating.

Frequency of treatment:

Daily, 7 days/week

Details on study schedule:

- Age at mating of the mated animals in the study: 12-13 weeks old (males) and 13-14 weeks old (females) at mating, as practical.

Dose / conc.:

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

Remarks:

Vehicle controls (Group 1)

Dose / conc.:

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

Remarks:

Group 2

Dose / conc.:

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

Remarks:

Group 3

Dose / conc.:

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

Remarks:

Group 4

No. of animals per sex per dose:

12/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on findings obtained in previous studies conducted by the Sponsor in other laboratories (i.e., 7- and 28-day repeated dose toxicity studies) in agreement with the Sponsor. Doses were selected with the aim of inducing toxic effects but no death or suffering at the highest dose and a NOAEL at the lowest dose.

Positive control:

Not applicable.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for morbidity and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: general clinical observations were conducted once a day, after treatment at approimately the same time. More detailed examinations were made once before the first exposure and at least weekly thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: on day 0 and at least weekly afterwards and at termination. Parent females were weighed on gestation Days GD 0, 7, 14 and 20 and on postpartal Days PPD 0 (within 24 hours after parturition) and 4 (before termination). Body weight of the female animals was additionally weighed on GD 10 and 17 in order to give accurate treatment volumes, but these data were not evaluated statistically.

FOOD CONSUMPTION: yes
- Time schedule: animal food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g on Day 7 then at least weekly

WATER CONSUMPTION: No

Oestrous cyclicity (parental animals):

Not evaluated.

Sperm parameters (parental animals):

Parameters examined in P male parental generation:
testis weight, epididymis weight, stages of spermatogenesis in the male gonads, histopathology of interstitial testicular cell structure

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no, pups sacrificed on PPD 4.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, pup body weight on PPD 0 and 4, body weight gain (per litter) between PPD 0-4, physical abnormalities

GROSS EXAMINATION OF DEAD PUPS:
Pups found dead were subjected to necropsy with macroscopic examination. Pups euthanized on PPD 4 were carefully examined at least externally for gross abnormalities.


ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: not performed

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: not performed

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All animals, following mating
- Maternal animals: All animals, on PPD 4 or, for females that showed no evidence of mating, 25-28 days after mating

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. The number of implantation sites and of corpora lutea was recorded.

HISTOPATHOLOGY: Detailed histological examination was performed on the testes, epididymides (total and cauda), seminal vesicles with coagulating glands, prostate, ovary, uterus (with and without cervix), vagina, brain and pituitary of the animals in the control and high dose groups, and all gross lesions from animals in all groups. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.


ORGAN WEIGHTS:
The following organ weights were determined: with a precision of 0.01 g: uterus (with and without cervix), vagina, testes, epididymides (total and cauda), prostate, seminal vesicles with coagulating glands, brain; with a precision of 0.001 g: ovaries, pituitary.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring was sacrificed on PPD 4.

GROSS NECROPSY
- Gross necropsy was conducted on pups that were found dead. Pups euthanized at PND 4 were carefully examined at least externally for gross abnormalities.

HISTOPATHOLOGY / ORGAN WEIGTHS: not examined.

Statistics:

The homogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to assess the significance of inter-group differences. Where significant result was obtained at Bartlett's test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed as required.

Reproductive indices:

Male mating index = 100% x (number of males with confirmed mating/total number of males cohabited)
Female mating index = 100% x (number of sperm-positive females/total number of females cohabited)
Male fertility index = 100% x (number of males impregnating a female/total number of males cohabited)
Female fertility index = 100% x (number of pregnant females/number of sperm-positive females)
Gestation index = 100% x (number of females with live born pups/number of pregnant females)

Offspring viability indices:

Survival index = 100% x (number of live pups at designated day/number of pups born)
Pre-implanation mortality loss = 100% x (number of corpora lutea - number of implantations)/number of corpora lutea
Post-implantation mortality loss = 100% x (number of implantations - number of liveborns) / number of implantations
Total mortality = 100% x (number of implantations - number of viable pups (PPD 4)/number of implantations
Sex ratio = 100 % x (number of pups examined - number of males)/number of pups examined

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

In males, there were no clinical signs of toxicity at any dose level.
No clinical signs of toxicity were observed in females at 25 mg/kg bw/day. At 150 mg/kg bw/day, 1 female had minor incidental clinical signs, such as scab or missing fur in the neck area, unrelated to treatment. At 1000 mg/kg bw/day, the female that died, starting from GD19, displayed clinical signs such as, but not limited to, vaginal bleeding and paleness (likely due to blood loss followed by anaemia) and activity decreased as of day 33, followed by uncoordination on day 37 and death on day 38. At necropsy, liver enlargement and dark red discoloration of the lungs were noted and were considered incidental or post-mortem changes. Placenta vaginal retention was observed, and 16 corpora lutea and 4 implantation sites were recorded, thus, a 75% pre-implantation mortality, with no live fetuses identified or liveborn pups. Based on the clinical signs and macroscopic findings recorded in this animal, and considering the isolated incidence within the group, its death was considered due to individual variability associated with abottion with placental retention, and not ascribed to test item systemic toxicity.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

In females, at the highest dose level 1 female died on day 38 (GD 24). No further mortalities were noted.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No treatment-related effects on body weight and body weight gain were noted at 25 and 150 mg/kg bw/day.
In the high dose 1000 mg/kg bw/day males, statistically significant, slightly lower than control body weight mean value was noted on Day 7, after one week of treatment. The mean body weight gain values were up to -49% compared to control, for example from Day 0 to Day 7 (pre-mating, p<0.01), with an overall mean from Day 0 to Day 28 of -19% lower than control (p<0.05).
In the high dose 1000 mg/kg bw/day females, the body weight gain mean values calculated from Days 0 to 14, GD0 to GD20, or PPD0 to PPD4 were up to -40% lower than control, however, with no statistical significance. Statistically significant lower body weight gain values (p<0.01) were observed between Days 0 to 7 (pre-mating) and GD 17 to 20, as well as prior to the scheduled necropsy on PND4.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

No treatment-related effect on food consumption was observed at 25 and 150 mg/kg bw/day.
At 1000 mglkg bw/day, both male and female animals had statistically lower mean food consumption between Day 0 to 7 (pre-mating), which correlated with the body weight values.
Lower feed intake was also noted in the high dose females between GD20 to parturition and during post-pattum period (PPDO to PPD4); however, the dose response was not consistent and these variations were considered individual variability due one female that died, and not a direct adverse effect of the test item.

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In the high-dose female that died, bilateral transmural suppurative inflammation with presence of bacteria in the uterus, mild luminar purulent exsudate with hemorrhages in both vagina and uterine cervix, were observed at histology evaluation and were considered to be the possible cause of death of this female.
There was no further evidence of test item-related histopathological findings. Minimal diffuse congestion in the liver and moderate diffuse congestion and/or hemorrhages in the lung correlated with the macroscopic observations were likely agonal. The follicular, luteal and interstitial compartments of the ovaty as well as epithelial capsule and stroma were similar histological structure in both control and high dose females. The primordial, secondary and tetiiary follicles and corpora lutea were also bilaterally present. Minor microscopic findings were recorded; based on the isolated occurrence they were regarded as incidental.
Common background microscopic changes seen across control and high dose males with low severity were noted in variety of reproductive organs including, testes, epididymides and prostate. In the testes, the spermatogenic cells, representing different phases of the development and differentiation of the spermatozoons as well as interstitial cell structure were similar in control and high dose males.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

In the testes, the spermatogenic cells, representing different phases of the development and differentiation of the spermatozoons as well as interstitial cell structure were similar in control and high Dose males.
Statistically higher absolute and/or relative seminal vesicle weights were observed at 150 and/or 1000 mg/kg bw/day, with an apparent dose response and slightly higher than the historical mean value (e.g. 2.84 g vs. 2.15 g), although still within the historical control range (1.28 g - 2.94 g) . In the absence of any macroscopic or microscopic findings, the significance of these variations was considered equivocal in relation to the test item treatment.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no differences considered significant between the control and test item-treated groups with regard to reproductive ability or in the mating, fertility and gestation indices.
In the male animals, the mating indices were 100 % in all groups, and the fertility indices were 100% in the low and mid dose groups and similar in the control and high dose animals (83%), due to 2 non-pregnant females in each group. This variation was considered incidental.
In the female groups, all animals paired, mated, with a mating index of 100%. Fertility indices were 100% at 25 and 150 mg/kg bw/day, and showed comparable values in the control and high dose groups, 83% and 91%, respectively. Gestation index was 100% at up to 150 mg/kg bw/day and 90% in the 1000 mg/kg bw/day group, however, this variation was not directly related to test item administration, but due to one individual female which had vaginal bleeding and placental retention, however, which was pregnant with 16 corpora lutea, 4 implantation sites, although with no live foetuses or liveborns were identified, on GD24 (Day 38).
The reproductive indices were within the normal ranges or considered individual variations unrelated to the test item administration.
Test item administration was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) generally occurred within up to 5 days of pairing (cohabitation), with the exception of one high dose female, which had a sperm positive vaginal smear after 7 days of mating.
No test item effect on the duration of pregnancy or abnormalities in the gestation outcome clearly ascribed to the treatment were observed. The mean duration of pregnancy was similar in the control and test item treated groups and varied from 22.7 days (22 to 23 days) in the controls, 22.83 days in the low 25 mg/kg bw/day (22 to 24 days) and in the mid 150 mg/kg bw/day dose groups (22 to 23 days), to 22.78 days (22 to 24 days) in the 1000 mg/kg bw/day high dose group, values comparable with the contemporaneous historical control data (gestation length: 22.40 ± 0.53 days, range: 22 to 23 days, n = 22). All the parturitions were normal, with the exception of one high dose female which showed parturition difficulties, with vaginal bleeding and placental retention, high pre-implantation mortality and no live foetuses or liveborns. Based on the isolated incidence, the observations noted in this female were considered incidental and not directly related to treatment, however, a possible effect of the test item on the uterine motility at 1000 mg/kg bw/day could not be excluded.
The number of corpora lutea and implantation sites were similar in the treated groups up to and including 1000 mg/kg bw/day, compared to females in the control groups.

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects on fertility at the highest tested dose level.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: general toxicity

Organ:

other: general toxicity

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the high-dose group, pups found dead were cold and/or not suckled. Similar clinical signs were noted in a few pups that survived to scheduled termination on PPD 4.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

There were no test item related adverse effects on the pre/post-implantation, post-natal or total mortality values (%) at up to and including 150 mg/kg bw/day. At 1000 mg/kg bw/day, higher than control postnatal mortality (33.68% vs. 0, p<0.01, with 8.44 vs. 14.40 mean number of live pups on PND4, p<0.05) and total mortality (pre-implantation, post-implantation, postnatal) (43.66% vs. 13.21%, p<0.05) were observed and were ascribed to the test item administration.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean litter weights on PND 0 and 4, pups body weight and body weight gain evaluated on PND 0 and 4, for all pups or per litter, showed statistically lower values compared to controls in the F1 generation following administration of the test item daily by oral gavage, to the parental generation, at the top dose of 1000 mg/kg bw/day.
No effects considered potentially adverse on the offspring weight or weight gain were noted at up to and including 150 mg/kg bw/day; minor variations, on occasion attaining statistically significance, were noted, however, the differences from control were subtle, with no dose response and not considered toxicologically significant.

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

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Out of pups found dead, necropsy was performed on 7 low, 1 mid and 20 high dose pups. The floating test was positive in 4/7, 1/1, and 17/20 pups from the low, mid- and high dose groups, respectively.
No macroscopic changes were seen in F1 offsprings euthanized at completion of the study on PND4.

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

body weight and weight gain

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

yes

Analytical determination of the test item concentrations in the dosing solutions:

The measured concentrations in percentage of nominal for low, mid- and high-dose groups varied in the rance of 98-102%, 97-105% and 99-105%, respectively.

Conclusions:

In the reproductive/developmental toxicity screening test with Wistar rats, conducted according to OECD guideline 421 and under GLP, the NOAEL for general toxicity in the parental generation was 150 mg/kg bw/day, based on decreased body weight and body weight gain in both sexes at the highest dose level. No adverse changes on fertility were noted at the highest tested dose of 1000 mg/kg bw/day, therefore this dose level is considered to be a NOAEL for reproductive toxicity. The NOAEL for developmental toxicity was 150 mg/kg bw/day, based on reduced viability, increased mortality and decreased body weight and body weight gain of pups at the highest tested dose level of 1000 mg/kg bw/day.

Executive summary:

In the reproductive/developmental toxicity screening test with Wistar rats, conducted according to OECD guideline 421 and under GLP, groups of 12 Wistar rats/sex/dose were administered the test item in water by oral gavage at dose levels of 0, 25, 150 and 1000 mg/kg bw/day. Females were allowed to litter normally and sacrificed on day 4 post-partum. Gross necropsy was conducted at the end of the treatment period. A histopathological examination was performed on the selected preserved organ and tissues of the parental animals of the control and high dose groups and on abnormal tissues of all animals. Gross necropsy was conducted on pups found dead; pups sacrificed on post-partum day 4, were examined at least externally for gross anomalities.

In the parental generation, there were no clinical signs related to treatment. At the highest dose level of 1000 mg/kg bw/day, one female died. In this female, vaginal bleeding, paleness and decreased activity were noted prior to death; gross necropsy showed 16 corpora lutea and 4 implantation sites (75% pre-implantation mortality), with no live fetuses or liveborn pups. Dark red discoloration of the non collapsed lungs, enlarged liver and placenta into vagina were found at necropsy. Bilateral transmural suppurative inflammation with presence of bacteria in the uterus, mild luminar purulent exsudate with hemorrhages in both vagina and uterine cervix, were observed at histology evaluation and were considered to be the possible cause of death of this female. Based on the clinical signs, pathology findings and isolated incidence within the group, its death was considered due to individual variability associated with abortion and placental retention, and not ascribed to test item systemic toxicity. Statistically significantly lower body gains were seen in both sexes at 1000 mg/kg bw/day, correlated with statistically lower mean food consumptions; lower body weights were also seen in males at day 7. No test item-related findings were observed at gross necropsy or histopathological examination. Based on the this, the NOAEL for general toxicity for parental animals was set at 150 mg/kg bw/day.

There were no differences considered significant between the control and test item-treated groups with regard to reproductive ability or in the mating, fertility and gestation indices. The treatment with the test item had no effects on the duration of the mating period and of the gestation. The number of corpora lutea and implantation sites were similar in the treated groups up to and including 1000 mg/kg bw/day, compared to females in the control groups. The highest dose level of 1000 mg/kg bw/day is considered the NOAEL for reproductive toxicity.

At 1000 mg/kg bw/day, higher than control preimplantation mortality, postnatal mortality and total mortality of the pups were observed and considered to be test item-related. Statistically higher than control numbers of pups found dead, cannibalized, not suckled, aud/or cold were recorded in the 1000 mg/kg bw/day group. No external abnormalities ascribed to test item administration were detected at the clinical or external macroscopic examinations of the pups. The number of viable pups on PND4 as well as pups survival indices on PND0 and PND4 were lower than control in the 1000 mg/kg bw/day group. The sex ratios were similar in the control and treated groups. The mean litter weights on PND 0 and 4, pups body weight and body weight gain evaluated on PND 0 and 4, for all pups or per litter, showed statistically lower values compared to controls in the F1 generation following administration of the test item to the parental generation at dose level of 1000 mg/kg bw/day. The NOAEL for developmental toxicity was considered to be 150 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

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 the reproductive/developmental toxicity screening test with Wistar rats, conducted according to OECD guideline 421 and under GLP, groups of 12 Wistar rats/sex/dose were administered the test item in water by oral gavage at dose levels of 0, 25, 150 and 1000 mg/kg bw/day. Females were allowed to litter normally and sacrificed on day 4 post-partum. Gross necropsy was conducted at the end of the treatment period. A histopathological examination was performed on the selected preserved organ and tissues of the parental animals of the control and high dose groups and on abnormal tissues of all animals. Gross necropsy was conducted on pups found dead; pups sacrificed on post-partum day 4, were examined at least externally for gross anomalities.

In the parental generation, there were no clinical signs related to treatment. At the highest dose level of 1000 mg/kg bw/day, one female died. In this female, vaginal bleeding, paleness and decreased activity were noted prior to death; gross necropsy showed 16 corpora lutea and 4 implantation sites (75% pre-implantation mortality), with no live fetuses or liveborn pups. Dark red discoloration of the non collapsed lungs, enlarged liver and placenta into vagina were found at necropsy. Bilateral transmural suppurative inflammation with presence of bacteria in the uterus, mild luminar purulent exsudate with hemorrhages in both vagina and uterine cervix, were observed at histology evaluation and were considered to be the possible cause of death of this female. Based on the clinical signs, pathology findings and isolated incidence within the group, its death was considered due to individual variability associated with abortion and placental retention, and not ascribed to test item systemic toxicity. Statistically significantly lower body gains were seen in both sexes at 1000 mg/kg bw/day, correlated with statistically lower mean food consumptions; lower body weights were also seen in males at day 7. No test item-related findings were observed at gross necropsy or histopathological examination. Based on the this, the NOAEL for general toxicity for parental animals was set at 150 mg/kg bw/day.

There were no differences considered significant between the control and test item-treated groups with regard to reproductive ability or in the mating, fertility and gestation indices. The treatment with the test item had no effects on the duration of the mating period and of the gestation. The number of corpora lutea and implantation sites were similar in the treated groups up to and including 1000 mg/kg bw/day, compared to females in the control groups. The highest dose level of 1000 mg/kg bw/day is considered the NOAEL for reproductive toxicity.

At 1000 mg/kg bw/day, higher than control preimplantation mortality, postnatal mortality and total mortality of the pups were observed and considered to be test item-related. Statistically higher than control numbers of pups found dead, cannibalized, not suckled, aud/or cold were recorded in the 1000 mg/kg bw/day group. No external abnormalities ascribed to test item administration were detected at the clinical or external macroscopic examinations of the pups. The number of viable pups on PND4 as well as pups survival indices on PND0 and PND4 were lower than control in the 1000 mg/kg bw/day group. The sex ratios were similar in the control and treated groups. The mean litter weights on PND 0 and 4, pups body weight and body weight gain evaluated on PND 0 and 4, for all pups or per litter, showed statistically lower values compared to controls in the F1 generation following administration of the test item to the parental generation at dose level of 1000 mg/kg bw/day. The NOAEL for developmental toxicity was considered to be 150 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

In the reproductive/developmental toxicity screening test with Wistar rats, conducted according to OECD guideline 421 and under GLP, the NOAEL for developmental toxicity was 150 mg/kg bw/day, based on reduced viability, increased mortality and decreased body weight and body weight gain of pups at the highest tested dose level of 1000 mg/kg bw/day. These effects occurred in the presence of maternal toxicity manifested as reduced body weight gains correlated with reduced food consumption.

In the available developmental toxicity study with rats, conducted according to OECD guideline 414, reduced fetal body weights were observed at dose levels causing reduced maternal body weights (500 mg/kg bw/day and above). In addition, an increase in the incidences of thymic remnant in the neck and a decrease in the number of sternebra ossification were observed in the 1000 mg/kg bw/day group as a secondary change associated with the growth retardation. As skeletal variations, an increase in the incidences of full and short supernumerary ribs was observed in the 1000 mg/kg bw/day group; however, no treated-related skeletal anomalies were observed in any live fetuses. Based on the observed effects, the NOAEL of 250 mg/kgbw/day was established for maternal general toxicity and embryo-fetal development.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 7, 2016 - September 20, 2016

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:

2001

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)

Route of administration:

oral: gavage

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

20 females/dose

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Key result

Abnormalities:

no effects observed

Localisation:

other: No abnormalities, only reduced body weight gain at two top doses

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: supernumerary rib

visceral/soft tissue: immune system

Description (incidence and severity):

Increased incidence of full and short supernumerary ribs and significantly lower number of sternebra ossification, and increased incidence of thymic remnant in the neck at 1000 mg/kg bw/day

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to maternal toxicity:

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

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

In the GLP-compliant OECD guideline 414 study with Crl:CD(SD) rats, decreased maternal body weight gain was observed starting from 500 mg/kg bw/day. No other treatment-related maternal effects were observed. In fetuses, decreased body weight was observed in live fetuses in the 500 and 1000 mg/kg bw/day groups. In addition, an increase in the incidences of thymic remnant in the neck and a decrease in the number of sternebra ossification were observed in the 1000 mg/kg bw/day group as a secondary change associated with the growth retardation. As skeletal variations, an increase in the incidences of full and short supernumerary ribs was observed in the 1000 mg/kg bw/day group. Based on these findings, the NOAEL for maternal and developmental toxicity was set at 250 mg/kg bw/day.

Executive summary:

In the GLP-compliant OECD guideline 414 study, groups of 20 female Crl:CD(SD) rats were administered the test substance in aqueous solution by oral gavage at dose levels of 0 (vehicle controls), 150, 250, 500 and 1000 mg/kg bg/day during days 7 -19 of gestation. The animals were euthanized on gestation day 20. No deaths occurred at any dose level and no clinical signs were observed in any group. Slight suppressed body weight gains were noted in dams in the 500 and 1000 mg/kg bw/day groups. No treatment-related effects were observed in food consumption, necropsy, or gravid uterus weights at any dose level. In fetuses, decreased body weight in live males and females fetuses was observed in the 500 and 1000 mg/kg bw/day groups. In addition, an increase in the incidences of thymic remnant in the

neck and a decrease in the number of sternebra ossification were observed in the 1000 mg/kg bw/day group as a secondary change associated with the growth retardation. As skeletal variations, an increase in the incidences of full and short supernumerary ribs was observed in the 1000 mg/kg bw/day group; however, no treated-related skeletal anomalies were observed in any live fetuses.

No treatment-related effects were observed in the number of corpora lutea, the number of implantations, pre-implantation loss index, post-implantation loss index, the number of live fetuses, or the sex ratio at any dose level. No treatment-related placental or external anomalies were observed at any dose level.

Based on the observed effects, the NOAEL of 250 mg/kgbw/day was established for maternal general toxicity and embryo-fetal development.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 January 2010 - 16 March 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study predates the most recent version of the guideline; thus, no evaluation of thyroid hormone levels were conducted.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD guideline 421 (Reproduction/Developmental Toxicity Screening Test)

Version / remarks:

27 July 1995

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

dd 2008-12-20

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature (15-25°C)

- Solubility and stability of the test substance in the solvent/vehicle: stable at 5±3°C pending use within 4 days (96 hours), or at room temperature pending use within 24 hours

Species:

rat

Strain:

Wistar

Remarks:

CRL:(WI) BR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 Sulzfeld
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 11-12 weeks old (females)
- Weight at study initiation: females: 237-266 g
- Fasting period before study: no
- Housing: group-housed up to 5 animals/sex/cage in type II and III polypropylene/polycarbonate cages, with the exception of the mating and gestation/delivery period, when they were paired or individually housed, respectively. Deep wood sawdus was provided for bedding and nesting.
- Diet: ssniff® SM RJM-Z+H "Autoclavable complete feed for rats and mice-breeding and maintenance" produced by ssniff Spezialdiaten GmbH, D-59494, Soest, Germany, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23.3
- Humidity (%): 24-50
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 28 January 2010 To: 16 March 2010

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations were prepared at the appropriate frequency and stored refrigerated at 5±3°C pending use within 4 days (96 hours), or at room temperature pending use within 24 hours, according to stability assessment results.

VEHICLE

- Concentration in vehicle: 2.5, 15 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of formulations (concentration and homogeneity) was performed in the analytical laboratory of the test facility. Top, middle and bottom samples were taken in duplicate for analysis of concentration and homogeneity from each of the test-item dosing solutions on 3 occasions, during the first and last weeks of treatment for both males and females. Similarly, one sample was taken in duplicate on each occasion from the control solution, for concentration measurements. The analyses were performed by HPLC.

Details on mating procedure:

- M/F ratio per cage: 1:1, mating of siblings avoided
- Length of cohabitation: up to 7 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- No further mating performed after 7 days of unsuccessful pairing
- After successful mating each pregnant female was caged individually

Duration of treatment / exposure:

Mated females: 14 days pre-mating, up to 7 days mating, throughout gestation and at least 4 days post-partum prior to necropsy.
Females showing no evidence of mating: 14 days pre-mating, up to 7 days mating, and 25-28 days after the last day of mating.

Frequency of treatment:

Daily, 7 days/week

Duration of test:

Females that mated: ca. 46 days (14 days pre-mating, up to 7 days mating, throughout gestation and at least 4 days post-partum prior to necropsy)
Females that showed no evidence of copulation: ca. 46 days (14 days pre-mating, up to 7 days mating, and 25-28 days after the last day of mating)

Dose / conc.:

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

Remarks:

Vehicle controls (Group 1)

Dose / conc.:

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

Remarks:

Group 2

Dose / conc.:

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

Remarks:

Group 3

Dose / conc.:

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

Remarks:

Group 4

No. of animals per sex per dose:

12/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on findings obtained in previous studies conducted by the Sponsor in other laboratories (i.e., 7- and 28-day repeated dose toxicity studies) in agreement with the Sponsor. Doses were selected with the aim of inducing toxic effects but no death or suffering at the highest dose and a NOAEL at the lowest dose.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for morbidity and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: general clinical observations were conducted once a day, after treatment at approimately the same time. More detailed examinations were made once before the first exposure and at least weekly thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: on day 0 and at least weekly afterwards and at termination. Parent females were weighed on gestation Days GD 0, 7, 14 and 20 and on postpartal Days PPD 0 (within 24 hours after parturition) and 4 (before termination). Body weight of the female animals was additionally weighed on GD 10 and 17 in order to give accurate treatment volumes, but these data were not evaluated statistically.

FOOD CONSUMPTION: yes
- Time schedule: animal food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g on Day 7 then at least weekly

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on post-partum day 4
- Organs examined: the following organ weights were determined in females: with a precision of 0.01 g: uterus (with and without cervix), vagina, brain; with a precision of 0.001 g: ovaries, pituitary. Detailed histological examination was performed on the ovary, uterus (with and without cervix), vagina, brain and pituitary of the animals in the control and high dose groups, and all gross lesions from animals in all groups. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.

Ovaries and uterine content:

The ovaries and uterus were examined after termination: Yes
Examinations included:
- Gravid uterus weight: no, females were allowed to litter naturally
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No

Fetal examinations:

- External examinations: yes, all per litter
- Soft tissue examinations: not specifically conducted, but gross necropsy was performed on all pups found dead and pups sacrified on postnatal day 4.
- Skeletal examinations: not specifically conducted, but gross necropsy was performed on all pups found dead and pups sacrified on postnatal day 4.
- Head examinations: not specifically conducted, but gross necropsy was performed on all pups found dead and pups sacrified on postnatal day 4.

Statistics:

The homogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to assess the significance of inter-group differences. Where significant result was obtained at Bartlett's test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed as required.

Indices:

The following offspring indices were calculated:
Survival index = 100% x (number of live pups at designated day/number of pups born)
Pre-implanation mortality loss = 100% x (number of corpora lutea - number of implantations)/number of corpora lutea
Post-implantation mortality loss = 100% x (number of implantations - number of liveborns) / number of implantations
Total mortality = 100% x (number of implantations - number of viable pups (PPD 4)/number of implantations
Sex ratio = 100 % x (number of pups examined - number of males)/number of pups examined

Historical control data:

Not included in the report.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs of toxicity were observed in females at 25 mg/kg bw/day. At 150 mg/kg bw/day, 1 female had minor incidental clinical signs, such as scab or missing fur in the neck area, unrelated to treatment. At 1000 mg/kg bw/day, the female that died, starting from GD19, displayed clinical signs such as, but not limited to, vaginal bleeding and paleness (likely due to blood loss followed by anaemia) and activity decreased as of day 33, followed by uncoordination on day 37 and death on day 38. At necropsy, liver enlargement and dark red discoloration of the lungs were noted and were considered incidental or post-mortem changes. Placenta vaginal retention was observed, and 16 corpora lutea and 4 implantation sites were recorded, thus, a 75% pre-implantation mortality, with no live fetuses identified or liveborn pups. Based on the clinical signs and macroscopic findings recorded in this animal, and considering the isolated incidence within the group, its death was considered due to individual variability associated with abottion with placental retention, and not ascribed to test item systemic toxicity.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

In females, at the highest dose level 1 female died on day 38 (GD 24). No further mortalities were noted.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No treatment-related effects on body weight and body weight gain were noted at 25 and 150 mg/kg bw/day. In the high dose 1000 mg/kg bw/day females, the body weight gain mean values calculated from Days 0 to 14, GD0 to GD20, or PPD0 to PPD4 were up to -40% lower than control, however, with no statistical significance. Statistically significant lower body weight gain values (p<0.01) were observed between Days 0 to 7 (pre-mating) and GD 17 to 20, as well as prior to the scheduled necropsy on PND4.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

No treatment-related effect on food consumption was observed at 25 and 150 mg/kg bw/day.
At 1000 mglkg bw/day, female animals had statistically lower mean food consumption between Day 0 to 7 (pre-mating), which correlated with the body weight values.
Lower feed intake was also noted in the high dose females between GD20 to parturition and during post-pattum period (PPDO to PPD4); however, the dose response was not consistent and these variations were considered individual variability due one female that died, and not a direct adverse effect of the test item.

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant variations were noted in the absolute or relative organ weights (e.g. brain, pituitary, ovaries), however, there was no consistent dose response, the variations were attributed to high control mean values (e.g. pituitary), or there were no correlated pathological and histopathological findings, thus, these variations were not considered toxicologically relevant or related to dose administration .

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related findings were observed at necropsy in parental generation. Renal pelvic dilatation, enlargement and pale area of the kidneys, dark discoloration/red of the lungs, enlarged spleen, ovarian cyst, and cyst in the adrenal gland were observed with low incidence in control and/or test-item treated female animals. These observations were regarded as incidental or terminal procedure-related.
In the high-dose female that died, dark red discoloration of the non collapsed lungs, enlarged liver and placenta into vagina were found.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In the high-dose female that died, bilateral transmural suppurative inflammation with presence of bacteria in the uterus, mild luminar purulent exsudate with hemorrhages in both vagina and uterine cervix, were observed at histology evaluation and were considered to be the possible cause of death of this female.
There was no further evidence of test item-related histopathological findings. Minimal diffuse congestion in the liver and moderate diffuse congestion and/or hemorrhages in the lung correlated with the macroscopic observations were likely agonal. The follicular, luteal and interstitial compartments of the ovaty as well as epithelial capsule and stroma were similar histological structure in both control and high dose females. The primordial, secondary and tetiiary follicles and corpora lutea were also bilaterally present. Minor microscopic findings were recorded; based on the isolated occurrence they were regarded as incidental.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Number of abortions:

no effects observed

Description (incidence and severity):

No test item effect on the abnormalities in the gestation outcome clearly ascribed to the treatment were observed.

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

There were no test item related adverse effects on the pre/post-implantation mortality values (%) at up to and including 150 mg/kg bw/day. At 1000 mg/kg bw/day, higher than control preimplantation mortality (15.65% vs. 5.78%, p<0.05) was observed and was ascribed to the test item administration. In the female that died, a 75% pre-implantation mortality (16 corpora lutea, 4 implantation sites) was observed, with no live fetuses or liveborn pups.

Total litter losses by resorption:

not examined

Early or late resorptions:

not examined

Dead fetuses:

no effects observed

Description (incidence and severity):

Intrauterine mortality was 20.03% in the high-dose group in comparison to 13.21% in controls; the effect was not statistically significant.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

The mean duration of pregnancy was similar in the control and test item treated groups and varied from 22.7 days (22 to 23 days) in the controls, 22.83 days in the low 25 mg/kg bw/day (22 to 24 days) and in the mid 150 mg/kg bw/day dose groups (22 to 23 days), to 22.78 days (22 to 24 days) in the 1000 mg/kg bw/day high dose group, values comparable with the contemporaneous historical control data (gestation length: 22.40 ± 0.53 days, range: 22 to 23 days, n = 22).

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Fertility indices were 100% at 25 and 150 mg/kg bw/day, and showed comparable values in the control and high dose groups, 83% and 91%, respectively.

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Key result

Abnormalities:

no effects observed

Localisation:

other: the only observed effects were lower body weight gain, correlated with lower substance intake

Fetal body weight changes:

not examined

Description (incidence and severity):

Fetal weights were not examined, as females were allowed to litter naturally.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Intrauterine mortality was 20.03% in the high-dose group in comparison to 13.21% in controls; the effect was not statistically significant.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex ratios were similar in the control and treated groups.

Changes in litter size and weights:

effects observed, treatment-related

Changes in postnatal survival:

effects observed, treatment-related

Description (incidence and severity):

The mean litter weights on PND 0 and 4, pups body weight and body weight gain evaluated on PND 0 and 4, for all pups or per litter, showed statistically lower values compared to controls in the F1 generation following administration of the test substance daily by oral gavage, to the parental generation, at the top dose of 1000 mg/kg bw/day.

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Description (incidence and severity):

Although skeletal malformations were not specifically examined, no macroscopic changes were seen in F1 offspring euthanized on PND 4 or found dead.

Visceral malformations:

no effects observed

Description (incidence and severity):

Although visceral malformations were not specifically examined, no macroscopic changes were seen in F1 offspring euthanized on PND 4 or found dead.

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

changes in litter size and weights

changes in postnatal survival

Key result

Abnormalities:

no effects observed

Localisation:

other: only effects on viability and body weights were noted.

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to maternal toxicity:

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

Dose response relationship:

yes

Relevant for humans:

yes

Analytical determination of the test item concentrations in the dosing solutions:

The measured concentrations in percentage of nominal for low, mid- and high-dose groups varied in the rance of 98-102%, 97-105% and 99-105%, respectively.

Conclusions:

In the reproductive/developmental toxicity screening test with Wistar rats, conducted according to OECD guideline 421 and under GLP, the NOAEL for general toxicity in the maternal generation was 150 mg/kg bw/day, based on decreased body weight and body weight gain at the highest dose level. The NOAEL for developmental toxicity was 150 mg/kg bw/day, based on reduced viability, increased mortality and decreased body weight and body weight gain of pups at the highest tested dose level of 1000 mg/kg bw/day.

Executive summary:

In the reproductive/developmental toxicity screening test with Wistar rats, conducted according to OECD guideline 421 and under GLP, groups of 12 Wistar rats/sex/dose were administered the test item in water by oral gavage at dose levels of 0, 25, 150 and 1000 mg/kg bw/day. Females were allowed to litter normally and sacrificed on day 4 post-partum. Gross necropsy was conducted at the end of the treatment period. A histopathological examination was performed on the selected preserved organ and tissues of the parental animals of the control and high dose groups and on abnormal tissues of all animals. Gross necropsy was conducted on pups found dead; pups sacrificed on post-partum day 4, were examined at least externally for gross anomalities.

In the parental generation, there were no clinical signs related to treatment. At the highest dose level of 1000 mg/kg bw/day, one female died. In this female, vaginal bleeding, paleness and decreased activity were noted prior to death; gross necropsy showed 16 corpora lutea and 4 implantation sites (75% pre-implantation mortality), with no live fetuses or liveborn pups. Dark red discoloration of the non collapsed lungs, enlarged liver and placenta into vagina were found at necropsy. Bilateral transmural suppurative inflammation with presence of bacteria in the uterus, mild luminar purulent exsudate with hemorrhages in both vagina and uterine cervix, were observed at histology evaluation and were considered to be the possible cause of death of this female. Based on the clinical signs, pathology findings and isolated incidence within the group, its death was considered due to individual variability associated with abortion and placental retention, and not ascribed to test item systemic toxicity. Statistically significantly lower body gains were seen in both sexes at 1000 mg/kg bw/day, correlated with statistically lower mean food consumptions; lower body weights were also seen in males at day 7. No test item-related findings were observed at gross necropsy or histopathological examination. Based on the this, the NOAEL for general toxicity for parental animals was set at 150 mg/kg bw/day. The treatment with the test item had no effects on the duration of the gestation, the number of corpora lutea and the number of implantation sites.

At 1000 mg/kg bw/day, higher than control preimplantation mortality, postnatal mortality and total mortality of the pups were observed and considered to be test item-related. Statistically higher than control numbers of pups found dead, cannibalized, not suckled, aud/or cold were recorded in the 1000 mg/kg bw/day group. No external abnormalities ascribed to test item administration were detected at the clinical or external macroscopic examinations of the pups. The number of viable pups on PND4 as well as pups survival indices on PND0 and PND4 were lower than control in the 1000 mg/kg bw/day group. The sex ratios were similar in the control and treated groups. The mean litter weights on PND 0 and 4, pups body weight and body weight gain evaluated on PND 0 and 4, for all pups or per litter, showed statistically lower values compared to controls in the F1 generation following administration of the test item to the parental generation at dose level of 1000 mg/kg bw/day. The NOAEL for developmental toxicity was considered to be 150 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subacute

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 the reproductive/developmental toxicity screening test with Wistar rats, conducted according to OECD guideline 421 and under GLP, groups of 12 Wistar rats/sex/dose were administered the test item in water by oral gavage at dose levels of 0, 25, 150 and 1000 mg/kg bw/day. Females were allowed to litter normally and sacrificed on day 4 post-partum. Gross necropsy was conducted at the end of the treatment period. A histopathological examination was performed on the selected preserved organ and tissues of the parental animals of the control and high dose groups and on abnormal tissues of all animals. Gross necropsy was conducted on pups found dead; pups sacrificed on post-partum day 4, were examined at least externally for gross anomalities.

In the parental generation, there were no clinical signs related to treatment. At the highest dose level of 1000 mg/kg bw/day, one female died. In this female, vaginal bleeding, paleness and decreased activity were noted prior to death; gross necropsy showed 16 corpora lutea and 4 implantation sites (75% pre-implantation mortality), with no live fetuses or liveborn pups. Dark red discoloration of the non collapsed lungs, enlarged liver and placenta into vagina were found at necropsy. Bilateral transmural suppurative inflammation with presence of bacteria in the uterus, mild luminar purulent exsudate with hemorrhages in both vagina and uterine cervix, were observed at histology evaluation and were considered to be the possible cause of death of this female. Based on the clinical signs, pathology findings and isolated incidence within the group, its death was considered due to individual variability associated with abortion and placental retention, and not ascribed to test item systemic toxicity. Statistically significantly lower body gains were seen in both sexes at 1000 mg/kg bw/day, correlated with statistically lower mean food consumptions; lower body weights were also seen in males at day 7. No test item-related findings were observed at gross necropsy or histopathological examination. Based on the this, the NOAEL for general toxicity for parental animals was set at 150 mg/kg bw/day. The treatment with the test item had no effects on the duration of the gestation, the number of corpora lutea and the number of implantation sites.

At 1000 mg/kg bw/day, higher than control preimplantation mortality, postnatal mortality and total mortality of the pups were observed and considered to be test item-related. Statistically higher than control numbers of pups found dead, cannibalized, not suckled, aud/or cold were recorded in the 1000 mg/kg bw/day group. No external abnormalities ascribed to test item administration were detected at the clinical or external macroscopic examinations of the pups. The number of viable pups on PND4 as well as pups survival indices on PND0 and PND4 were lower than control in the 1000 mg/kg bw/day group. The sex ratios were similar in the control and treated groups. The mean litter weights on PND 0 and 4, pups body weight and body weight gain evaluated on PND 0 and 4, for all pups or per litter, showed statistically lower values compared to controls in the F1 generation following administration of the test item to the parental generation at dose level of 1000 mg/kg bw/day. The NOAEL for developmental toxicity was considered to be 150 mg/kg bw/day.

In the GLP-compliant OECD guideline 414 study, groups of 20 female Crl:CD(SD) rats were administered the test substance in aqueous solution by oral gavage at dose levels of 0 (vehicle controls), 150, 250, 500 and 1000 mg/kg bg/day during days 7 -19 of gestation. The animals were euthanized on gestation day 20. No deaths occurred at any dose level and no clinical signs were observed in any group. Slight suppressed body weight gains were noted in dams in the 500 and 1000 mg/kg bw/day groups. No treatment-related effects were observed in food consumption, necropsy, or gravid uterus weights at any dose level.

In fetuses, decreased body weight in live male and female fetuses was observed in the 500 and 1000 mg/kg bw/day groups. In addition, an increase in the incidences of thymic remnant in the neck and a decrease in the number of sternebra ossification were observed in the 1000 mg/kg bw/day group as a secondary change associated with the growth retardation. As skeletal variations, an increase in the incidences of full and short supernumerary ribs was observed in the 1000 mg/kg bw/day group; however, no treated-related skeletal anomalies were observed in any live fetuses.

No treatment-related effects were observed in the number of corpora lutea, the number of implantations, pre-implantation loss index, post-implantation loss index, the number of live fetuses, or the sex ratio at any dose level. No treatment-related placental or external anomalies were observed at any dose level. Based on the observed effects, the NOAEL of 250 mg/kgbw/day was established for maternal general toxicity and embryo-fetal development.

Toxicity to reproduction: other studies

Description of key information

No further studies are available.

Mode of Action Analysis / Human Relevance Framework

In the available reproductive/developmental toxicity screening study and in the developmental toxicity study with rats, adverse effects on the offspring (increased preimplantation mortality, postnatal mortality, reduced number of viable pups and reduced fetal/pup body weights) were seen at dose levels causing maternal toxicity (reduced body weights and body weight gains, reduced food consumption). As these adverse effects do not reflect a specific mode of action which may be specific to the test species, they should be regarded as relevant for humans.

Justification for classification or non-classification

For the reproductive endpoints an OECD 421 study (Reproduction Developmental Toxicity Screening Test) and an OECD 414 study (Prenatal Developmental Toxicity Screening) are available.

The dose levels applied in the OECD 421 test were 0, 25, 150 and 1000 mg/kg bw/d. Based on findings of reduced body weight and weight gain, a NOAEL of 150 mg/kg bw/d is determined for general toxicity in the parental generation. No effects on fertility were observed at any of the dose levels (NOAEL >= 1000 mg/kg bw/d). For developmental toxicity, the NOAEL was set to 150 mg/kg bw/d based on findings of pup mortality and decreased viability, decreased mean litter weight, decreased pup body weight and body weight gain at the highest dose level.

Similar observations were made in the OECD 414 test. The dose levels in this test were 0, 150, 250, 500 and 1000 mg/kg bw/d. The NOAEL for general toxicity to the maternal animals is 250 mg/kg bw/d based on reduced body weight gains at the higher dose groups. For the fetuses, the developmental NOAEL was set to 250 mg/kg bw/d based on findings indicative of fetal growth retardation.

Based on the findings in these two studies, the need for a classification as Repro Cat. 2 needs to be assessed in line with the classification criteria described in section 3.7.2.1.1 of CLP.

In both studies, the observed reproductive effects occur together with certain levels of maternal toxicity. In section 3.7.2.2 of CLP, it is stated that ‘classification as a reproductive toxicant is intended to be used for substances which have an intrinsic, specific property to produce an adverse effect on reproduction and substances shall not be so classified if such an effect is produced solely as a non-specific secondary consequence of other toxic effects’. From the available repeated dose toxicity studies, it is known that at a dose level of 100 mg/kg bw/d and above, adverse effects on the liver occur, which lead to impaired homeostatis and increased cholesterol levels in the blood. Effects on the kidneys are also described.

As a consequence, in the reproductive toxicity tests substantial levels of maternal toxicity are expected at the dose levels of 150 mg/kg bw/d and above. Even though the reproductive studies did not include in-depth assessment of the parameters relevant to confirm toxicity in the dams (e.g. liver and kidney histopathology and clinical chemistry examinations), it is reasonable to believe that these maternal toxicity symptoms (e.g. elevated cholesterol levels) will have been present in the dams, and that they will have an effect on the development of the pup. A causal relationship between the maternal toxicity and developmental toxicity is therefore expected. The registrant thus considers the available information inconclusive to support classification of the substance as a Repro Cat. 2.