Tris(2-hydroxyethyl)ammonium salts of tall-oil fatty acids

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS: Wistar Crl:WI rats
- Source:Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 Sulzfeld
- Age at study initiation: 10 - 11 weeks
- Weight at study initiation: Males: 340 g – 395 g, Females: 230 g - 265 g
- Fasting period before study: overnight prior to treatment
- Housing: 5 animals of the same sex and group/cage with the exception of the mating and gestation/delivery period, when they
were paired or individually housed, respectively. (cage: Type II and/or III polycarbonate)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20,1-25
- Humidity (%): 36-70
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

VEHICLE
- Concentration in vehicle: 20, 60, 200 mg/ml
- Amount of vehicle (if gavage): 5 mL/kg bw

According to the results of a stability study, the test material was stable in Propylene glycol in concentration range of 20-200 mg/mL for 24 hours at room temperature (RT, 15-30ºC) and for up to three days when stored refrigerated at 2-8ºC (protected from light) and the homogeneity of the formulations was satisfactory.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: for up to 6 days
- After successful mating each pregnant female was caged individually
- Mating of siblings was avoided.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of WS400102 formulations for concentration and homogeneity was performed using validated HPLC-UV method (CiToxLAB study code 11/350-316AN). The concentration analysis was performed on 3 occasions, during the first, fourth and last weeks of the treatment period. Recovery of WS400102 from propylene glycol was in the range from 97 to 105%.

Duration of treatment / exposure:

Main males: 35 days (14 days pre-mating, 14 days mating/post-mating period followed by an additional week)
Main females: ca. 47 days (14 days pre-mating, for up to 7 days mating period, through gestation til PPD 4)
[Satellite females (nulliparous and nonpregnant): 35 days]

Frequency of treatment:

daily, 7 days/week

Details on study schedule:

- Age at mating of the mated animals in the study: 12-13 weeks

Dose / conc.:

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

Dose / conc.:

300 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:

Main groups: 12 animals/sex/dose
[Satellite group (20 femal rats): 5 animals/dose]
Offspring were not dosed.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Based on available data and information from previous experimental work, including the results of a preliminary dose range finding study in the rat (Endpoint study report 7.5.1 Dose Range Finding Study 7 days rat_WS400102) , the dose levels selected for this study were 100, 300 and 1000 mg/kg bw/day.

This study was conducted to examine both repeated dose toxicity and reproductive/developmental toxicity as an OECD screening combined study (OECD 422 test guideline). Therefore, animals initially entering the study were divided into toxicity subgroup animals (Satellite females) and reproductive subgroup animals (Main females and males), whereby 5 of the 12 Main males (used for pairing) per dose group formed the toxicity male Subgroup A.

Positive control:

No

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS
- Time schedule: twice daily
All animals were monitored for pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality.
Delivery process was observed as carefully as possible. Dams were observed to record whether they form a nest from the bedding material and cover their new-borns or not.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least weekly (observations in a standard arena)

BODY WEIGHT: Yes
- Time schedule: Parent females were weighed on gestation Days GD 0, 7, 14 and 20 and on postpartal Days PPD0 (within 24 hours after parturition), and PPD5 (before termination).
Parent males were weighed on Day 0 and at least weekly.

Sperm parameters (parental animals):

Parameters examined in male parental generations, all males (12/dose):
testes, epididymides (total and cauda), prostate, seminal vesicles with coagulating glands.
Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Litter observations:

STANDARDISATION OF LITTERS Not performed. The study ended on Lactation Day 4.

The following parameters were examined in F1- offspring:
number and sex of pups, stillbirths, live births; postnatal mortality; presence of gross anomalies, weight gain (PPD0-PPD4), physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes; for external abnormalities, any pups showing abnormalities in structure or behaviour were subjected to necropsy with macroscopic examination.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals on Day 35.
- Maternal animals: All surviving animals on Day 5 of lactation (PND5).

GROSS NECROPSY
- Gross necropsy consisted of external examinations including the cervical, thoracic, and abdominal viscera.
- Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea were recorded in the Main females as applicable.

ORGAN WEIGHTS
Weight of the following organs of all adult animals 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

HISTOPATHOLOGY :
Detailed histological examinations was performed in all main adults of control and high dose groups.
Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.
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.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring on Day 4:
Pups were carefully examined at least externally for gross abnormalities. Any pups showing abnormalities in structure or behaviour were subjected to necropsy with macroscopic examination. The probable cause of death of dead pups were recorded if it can be identified, e.g. cannabilism.

Macroscopic examination included assessment of the presence of milk in the stomach, where possible.

Statistics:

The statistical evaluation of appropriate data (marked † below) was performed with the statistical program package SPSS PC+4.0. 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 access the significance of inter-group differences. Getting significant result 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 feasible.

Reproductive indices:

Formulas for Calculation of Mating and Fertility Indices
Male Mating Index: Number of males with confirmed mating : Total Number of males cohabited x 100
Female Mating Index: Number of sperm-positive females : Total Number of females cohabited x 100
Male Fertility Index: Number of males impregnating a female : Total Number of males cohabited x 100
Female Fertility Index: Number of pregnant females : Number of sperm-positive females x 100
Gestation Index: Number of females with live born pups : Number of pregnant females x 100

Offspring viability indices:

Formulas for Calculation of Pups’ Mortality and Sex Ratio Indices
Survival Index: Number of live pups (at designated time) : Number of pups born x 100
Pre-implantation mortality: (Number of Corpora lutea − Number of Implantations) : Number of Corpora lutea x 100
Intrauterine mortality: (Number of implantations - Number of liveborns) : Number of implantations x 100
Total mortality: (Number of implantations - Number of viable pups (d4)) : Number of implantations x 100
Post-natal mortality: (Number of viable pups (d0) - Number of viable pups (d4)) : Number of viable pups (d0) x 100
Sex ratio: (Number of pups exa min ed − Number of males) : Number of pups examined x 100

Clinical signs:

no effects observed

Mortality:

no mortality observed

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:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Findings noted in the liver of males at 1000 mg/kg bw/day (High dose). Slight effects in kidneys of females.

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

see Table on Reproductive Performance attached in attached background material

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There was no mortality during the study.
There were no clinical signs related to treatment.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
There was no adverse effect of test material on body weight or body weight gain. Body weights were comparable to the control in all treated groups.
There was no adverse effect of treatment on food consumption.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There was no effect of treatment on the oestrus cycle or reproductive parameters.There were no differences between the Control and test material-treated groups with regard to reproductive ability or in the mating or gestation indices, or effects considered adverse or toxicologically significant in correlation with WS400102 administration.

The mating indices were 100% in all groups, while fertility indices were 92% in control and Mid groups, 83% in Low group and 75% in High dose animals.
There is no statistical difference to control, but a relationship with treatment cannot be excluded. Any differences are considered to be probably related to maternal toxicity.
WS400102 was considered to have no impact on the duration of the mating period. Successful coitus generally occurred within up to 6 days of pairing.
The mean duration of pregnancy was similar in the control and test material treated groups and varied between 22 and 23 days. The differences were considered as individual animal variation.

The gestation index was 100% in control, Low and Mid groups and 89% at 1000 mg/kg bw/day (High dose). In the High dose group, the slightly lower gestation index value was due to one female, pregnant with two implantation sites but not delivered. However, values are comparable with concurrent control data in Wistar rats. (see Table 1 "Any other information on results incl. tables")
All the parturitions were normal.

Slightly increased pre-implantation, total intrauterine and postnatal mortality values in the High dose group were observed.These small differences were considered to be probably secondary to maternal toxicity.

ORGAN WEIGHTS, GROSS PATHOLOGY, HISTOPATHOLOGY (PARENTAL ANIMALS)
Animals had slightly increased liver weights (by approximately 23-26% in males and 16-19% in females) accompanied by a minimal/mild changes in males in the form of diffuse hepatocellular vacuolation of all lobes (6/9 males) and centrilobular hypertrophy (4/9 males).
Females had histological minimal changes in kidneys in the form of bilateral vacuolation and presence of cytoplasmic eosinophilic granules in the cortical tubules (2/5 females).

No test material-related microscopic changes were noted in the reproductive organs at a dose level of 1000 mg/kg bw. Histopathological evaluation of the male gonads as well as testicular interstitial cell structure, the spermatogenic cells representing different phases of the development and differentiation of the spermatozoons were similar in Control and High Dose males. The follicular, luteal and interstitial compartments of the ovary as well as epithelial capsule and stroma were similar histological structure in both Control and High Dose females.
Randomly distributed changes across control and treated groups were regarded as incidental or common background.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 300 - <= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: slight changes in haematology, clinical chemistry, kidney (females only), liver (males only) in high dose animals

Organ:

kidney

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

see Tables on F1 generations attached in attached background material

Body weight and weight changes:

no effects observed

Description (incidence and severity):

see Table on body weight of F1 generation attached in attached background material

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Description (incidence and severity):

No abnormalities were observed at external examination.

Histopathological findings:

not examined

Other effects:

no effects observed

VIABILITY/CLINICAL SIGNS (OFFSPRING)
WS400102 administered to parental generation at up to 1000 mg/kg bw/day did not lead to mortality or any adverse effects considered related to treatment or toxicologically significant in the F1 generation. No abnormal behaviour of the pups was noted. No external abnormalities ascribed to treatment were detected at the clinical or external macroscopic examinations of the pups.
The number of viable pups on PND4 was slightly lower at 1000 mg/kg bw/day, when compared to control (p<0.05), which was in accordance with slightly lower number of live-born pups noted on PND0.

The survival indices of pups on PND0 and PND4 were comparable to control values at up to and including 1000 mg/kg bw/day. The incidence of mortality was negligible.
The sex ratios were similar in the Control and treated groups.

BODY WEIGHT (OFFSPRING)
There was no adverse effect of treatment on the offspring body weight or body weight gain.
Mean body weights values evaluated for all pups or on litter basis were similar in all treated groups.
Compared to control, slightly lower mean overall body weight gain values (PND0-4) were noted at 1000 mg/kg. The differences were in the range of 5% (litter basis) and 7% (all pups) and the differences attained statistical significance for all pups basis (p<0.05).

GROSS PATHOLOGY
No abnormalities in structure or behaviour were noted, therefore no necropsy with macroscopic examination was necessary.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: NOAEL = highest dose tested, administered to the parental animals, offspring were not dosed, F1 observations up to 4 days of age.

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

In conclusion, in this study - OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test) - daily oral gavage administration of WS400102 to Wistar rats at dose levels of 100, 300 and 1000 mg/kg bw/day was associated with week signs of reproduction toxicity in the High dose group and the NOAEL was considered to be 300 mg/kg bw/day. These effects were considered to be secondary to parental toxicity.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The reproduction toxicity screening test is adequate for an initial assessment of effects on fertility; it is of high quality and reliability.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The UVCB substance WS400102 to the biggest part is a physical mixture / salt of tall oil fatty acids and (technical) triethanolamine (TEA); only a small portion reacts forming monoesters. The technical quality of TEA contains up to 15% diethanolamine (DEA). DEA is known for its toxicity towards haematological parameters, liver and kidney in rats (Melnick et al, 1994). A NOAEL for haematological changes could not be derived in this study. The LOAEL after 90 day oral exposure amounted to 15 mg and 25 mg/kg body weight/day in females and males, respectively.

The highest dose applied in the combined repeated dose toxicity study with reproduction / developmental toxicity screening was 1000 mg/kg/day. This dose of WS400102 corresponds to up to 50 mg DEA/kg body weight/day according to the chemical composition of the UVCB substance. The effects observed in the parental animals of the high dose group after 35 days of exposure to WS400102 focused on changes in haematology, clinical chemistry parameters as well as slight effects in liver and kidney. These effects qualitatively and quantitatively correlated well with the effects observed in the 90 day study with DEA. Thus, it is very likely that the slight effects observed in the present study on reproductive parameters in high dose females are due to the toxicity of DEA.

DEA is classified (Regulation 1272/2008) STOT RE2 for specific organ toxicity next to acute toxicity, i.e. oral (cat. 4), skin irritation, eye damage, but not for reproduction toxicity. The other two substances used in the manufacture of WS400102, i.e. TEA and tall oil fatty acids, are not classified at all. Also esters that may be formed at low levels during manufacture of WS400102 are not toxic to reproduction as was shown for another substance produced from the same starting materials (CAS 67784 -78 -5).

In sum, there is no need for classification of WS400102 for reproductive toxicity.

Melnick, R.L., Mahler, J., Bucher, J.R., Thompson, M., Hejtmancik, M., Ryan, M.J., and Mezza, L.E. (1994): Toxicity of diethanolamine. 1. Drinking water and topical application exposures in F344 rats. In: J. Appl. Toxicol. 14, pp. 1 – 9.

Short description of key information:
In the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD 422) next to the parameters determined to examine toxicological effects in general, the reproductive performance and indices were monitored in the adult main animals, such as gonadal function, mating behaviour, conception, pregnancy, parturition and postpartum/lactation period.

Results: There was no effect of treatment on the oestrus cycle or reproductive parameters like reproductive ability, mating period, gestation and fertility indices as well as on parturition and post-partal period. Slightly increased pre-implantation, total intrauterine and postnatal mortality values and slightly lower number of born pups as well as slightly lower body weight gain of pups were observed in animals of the high dose group (1000 mg/kg/day). These small, statistically not significant differences were considered to be probably secondary to maternal toxicity.

Justification for selection of Effect on fertility via oral route:
Well performed screening study. The slight effects observed are considered secondary to maternal toxicity and not indicative of reproductive toxicity.

Effects on developmental toxicity

Description of key information

The Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD 422) included a development toxicity-screening test, intended to provide information on possible effects on the development of the F1 offspring from conception to Day 4 post-partum.
Offspring examinations: clinical signs, body weight, body weight gain, number of live births and of viable pups per litter on postnatal Days 0 and 4, sex ratio.
Results: Adverse effects on the maternal (and male) animals in the high dose group included  changes in haematology and clinical chemistry parameters. Slightly increased liver weights, histological minimal changes in kidneys (females only) were observed.
There were no adverse effects on the F1 offspring viability, clinical signs or development. 

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Remarks:

from OECD 422 study with reproduction / developmental toxicity screening

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS: Wistar Crl:WI rats
- Source:Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 Sulzfeld
- Age at study initiation: 10 - 11 weeks
- Weight at study initiation: Males: 340 g – 395 g, Females: 230 g - 265 g
- Fasting period before study: overnight prior to treatment
- Housing: 5 animals of the same sex and group/cage with the exception of the mating and gestation/delivery period, when they
were paired or individually housed, respectively. (cage: Type II and/or III polycarbonate)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20,1-25
- Humidity (%): 36-70
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

VEHICLE
- Concentration in vehicle: 20, 60, 200 mg/ml
- Amount of vehicle (if gavage): 5 mL/kg bw

According to the results of a stability study, the test material was stable in Propylene glycol in concentration range of 20-200 mg/mL for 24 hours at room temperature (RT, 15-30ºC) and for up to three days when stored refrigerated at 2-8ºC (protected from light) and the homogeneity of the formulations was satisfactory

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of WS400102 formulations for concentration and homogeneity was performed using validated HPLC-UV method (CiToxLAB study code 11/350-316AN). The concentration analysis was performed on 3 occasions, during the first, fourth and last weeks of the treatment period. Recovery of WS400102 from propylene glycol was in the range from 97 to 105%.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: for up to 6 days
- After successful mating each pregnant female was caged individually
- Mating of siblings was avoided.

Duration of treatment / exposure:

Main males: 35 days (14 days pre-mating, 14 days mating/post-mating period followed by an additional week)
Main females: ca. 47 days (14 days pre-mating, for up to 7 days mating period, through gestation til PPD 4)
[Satellite females (nulliparous and nonpregnant): 35 days]

Frequency of treatment:

daily, 7 days/week

Dose / conc.:

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

Dose / conc.:

300 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:

Main groups: 12 animals/sex/dose
[Satellite group (20 femal rats): 5 animals/dose]
Offspring were not dosed.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Based on available data and information from previous experimental work, including the results of a preliminary dose range finding study in the rat (Endpoint study report 7.5.1 Dose Range Finding Study 7 days rat_WS400102) , the dose levels selected for this study were 100, 300 and 1000 mg/kg bw/day.

This study was conducted to examine both repeated dose toxicity and reproductive/developmental toxicity as an OECD screening combined study (OECD 422 test guideline). Therefore, animals initially entering the study were divided into toxicity subgroup animals (Satellite females) and reproductive subgroup animals (Main females and males), whereby 5 of the 12 Main males (used for pairing) per dose group formed the toxicity male Subgroup A.

Maternal examinations:

CAGE SIDE OBSERVATIONS
- Time schedule: twice daily
All animals were monitored for pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality.
Delivery process was observed as carefully as possible. Dams were observed to record whether they form a nest from the bedding material and cover their new-borns or not.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least weekly (observations in a standard arena)

BODY WEIGHT: Yes
- Time schedule: Maternal animals were weighed on gestation Days GD 0, 7, 14 and 20 and on postpartal Days PPD0 (within 24 hours after parturition), and PPD5 (before termination).

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on Day 5 of lactation (PND5)
- Organs examined: uterus, vagina, ovaries

HISTOPATHOLOGY :
Detailed histological examinations was performed in all maternal animals of control and high dose groups 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 uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes (with and wtihout cervix)
- Number of corpora lutea: Yes
- Number of implantations: Yes

Post implantation survival was determined.

In addition, gestation length (time elapsing between detection of mating and commencement of parturition) was recorded.

Fetal examinations:

- External examinations: from PPD0 to PPD4, all pubs per litter
parameters observed: number and sex of pups, stillbirths, live births; postnatal mortality; presence of gross anomalies, weight gain (PPD0-PPD4), physical or behavioural abnormalities

Statistics:

The statistical evaluation of appropriate data (marked † below) was performed with the statistical program package SPSS PC+4.0. 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 access the significance of inter-group differences. Getting significant result 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 feasible.

Indices:

Female Mating Index: Number of sperm-positive females : Total Number of females cohabited x 100
Female Fertility Index: Number of pregnant females : Number of sperm-positive females x 100
Gestation Index: Number of females with live born pups : Number of pregnant females x 100

Formulas for Calculation of Pups’ Mortality and Sex Ratio Indices
Survival Index: Number of live pups (at designated time) : Number of pups born x 100
Pre-implantation mortality: (Number of Corpora lutea − Number of Implantations) : Number of Corpora lutea x 100
Intrauterine mortality: (Number of implantations - Number of liveborns) : Number of implantations x 100
Total mortality: (Number of implantations - Number of viable pups (d4)) : Number of implantations x 100
Post-natal mortality: (Number of viable pups (d0) - Number of viable pups (d4)) : Number of viable pups (d0) x 100
Sex ratio: (Number of pups exa min ed − Number of males) : Number of pups examined x 100

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Behaviour (functional findings):

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

in high dose females of the OECD 422 study microscopic findings were observed in kidney

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

see Tables on F1 generation attached in attached background material

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

see Tables on F1 generation attached in attached background material

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

see Tables on F1 generation attached in attached background material

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

see Tables on F1 generation attached in attached background material

Other effects:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: In High dose group: changes in haematology, in some parameters of clinical chemistry, slightly increased liver weight, histological minimal changes in kidneys

Details on maternal toxic effects:
In the High dose group (1000 mg): Changes in haematology (decrease of 8-12% in erythrocyte count, hemoglobin concentration and hematocrit value) and clinical chemistry parameters (increase in albumin concentration and albumin to globulin ration, higher calcium, sodium and chloride concentrations). Animals had slightly increased liver weights (16-19%) changes. Females had histological minimal changes in kidneys in the form of bilateral vacuolation and presence of cytoplasmic eosinophilic granules in the cortical tubules (2/5 females).
Slightly increased pre-implantation, total intrauterine and postnatal mortality values and slightly lower number of born pups as well as slightly lower body weight gain of pups were observed. These small differences were considered to be probably secondary to maternal toxicity.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

>= 300 - <= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Fetal body weight changes:

no effects observed

Description (incidence and severity):

see Tables on F1 generation attached in attached background material

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

see Tables on F1 generation attached in attached background material

Changes in sex ratio:

no effects observed

Description (incidence and severity):

see Tables on F1 generation attached in attached background material

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

see Tables on F1 generation attached in attached background material

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

see Tables on F1 generation attached in attached background material

External malformations:

no effects observed

Description (incidence and severity):

see Tables on F1 generation attached in attached background material

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No external abnormalities ascribed to treatment were detected at the clinical or external macroscopic examinations of the pups. At 300 mg/kg in one male pup aplasia of the tail was detected without any internal abnormality. The incidence of this finding was low, within the physiological range expected in the population of Wistar rats and considered without toxicological significance, or to reflect a test item or adverse effect.

The survival indices of pups on PND0 and PND4 were comparable to control values at up to and including 1000 mg/kg bw/day. The incidence of mortality was negligible and was 1 of 160 in control, 1 of 156 in Low dose, 6 of 155 in Mid dose and 3 of 89 in High dose. Three of 6 dead pups in the Mid dose and 3 of 3 dead pups in the High dose originated from one litter. All of these pups were lost on Day PND1, except one pup of Mid dose (PND4) and control (PND2). These pups were probably cannibalized.The sex ratios were similar in the Control and treated groups.

There was no adverse effect of treatment on the offspring body weight or body weight gain. Slightly higher mean body weights values in the Low dose group and slightly lower mean overall body weight gain values (PND0-4) in the High dose group were regarded as individual, biological variability, without
toxicological significance.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

WS400102 administered to parental generation at up to 1000 mg/kg bw/day did not lead to mortality or any adverse effects considered related to treatment or toxicologically significant in the F1 generation.

Effect on developmental toxicity: 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:

The development toxicity screening test is is adequate for an initial assessment of effects on development toxicity; it is of high quality and reliability.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Following the results of the available studies classification of WS400102 regarding reproductive or developmental toxicity is not warranted according to European classification rules [ REGULATION (EC) 1272/2008].