Aluminium tridiethylphosphinate

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

The registration substance did not induce any toxicity in the EOGRT up to the highest dose of 1000 mg/kg/day.

Link to relevant study records

Reference

Endpoint:

extended one-generation reproductive toxicity - with developmental neurotoxicity (Cohorts 1A, 1B without extension, 2A and 2B)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Deviations:

yes

Remarks:

Without breeding of 1A and 1B

GLP compliance:

yes (incl. QA statement)

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

- Premating exposure duration for parental (P0) animals:
10 weeks
- Basis for dose level selection:
Based on previously performed OECD 421 and OCED 414 (refer to Section 7.8.1 and 7.8.2 of the IUCLID)
- Inclusion/exclusion of extension of Cohort 1B: Cohort 1 B is to be separated as reserve if a second generation will be necessary (depends on outcome of cohort 1A).

- Exclusion of developmental neurotoxicity Cohorts 2A and 2B:
Reason: In order to assess the systemic, internal uptake of aluminium or phosphorus an in vitro toxicokinetic bioassesability study was performed in 2010. (Bioaccessibility of Aluminium and Phosphorus from EPAL during transit through the TNO dynamic gastro intestinal model simulating the upper GI tract under fasting conditions (TIM-1 system)). This study is provided in Section 7.1.1 of the IUCLID. The solubility and availability for absorption through the gut wall (bioaccessibility) of the Phosphorus and Aluminium component of the test substance are determined in a model system that simulates the dynamic conditions in the gastro-intestinal tract (TIM-1). The TNO in vitro gastrointestinal models simulate in high degree the successive dynamic processes in the stomach and the small intestine (TIM-1 system). This system is a unique tool to study the stability, release, dissolution, absorbtion and bioconversion of nutrients, chemicals, bioactive compounds and pharmaceuticals in the gastrointestinal (GI) tract. The model has been validated for the simulation of physiological conditions within the lumen of the GI tract in the food and nutrition area as well as in the pharmaceutical area. The results obtained in the TIM system showed very good resemblance with the results obtained in studies with humans and animals for drugs and formulations.
The endogenous intake of aluminium and phosphorus was negligible compared to the doses coming from the substance. Bioaccessible aluminium and phosphorus from the endogenous intake were 0 mg and 11.2 mg, respectively. After administration of 678.4 mg aluminium from the substance, 0.1% of the administered aluminium dose (0.7 mg aluminium and corresponding to 0.008% of the complete substance dose) was bioaccessible, 50.6% was emptied from the ileum compartment and 47.4% was retained in the compartments of the TIM-system as non-bioaccessible fractions. For phosphorus, the bioaccessible fraction from a 2344 mg dose was 26.3% (617.8 mg). From this dose was 39.0% emptied from the ilem and 39.2% was recovered from the compartments at the end of the experiment. These fractions can be regarded as non-bioaccessible.
There is no indication for neurotoxicity seen in the provided data, e.g. OECD 407 in section 7.5.2. Neurotoxicological measurements including assessment of sensory function, number of rearings, motor activity, forelimb and hind limb grip strength as well as landing foot spread were not influenced by the administration of the test compound in all groups.

- Exclusion of developmental immunotoxicity Cohort 3
Reason: There is no indication for any immunotoxicological effects given in available studies, e.g. OECD 407 study. The hematological examinations revealed statistically significant decreased MCV values and increased leukocyte counts in females of the high dose group (i.e. 1000 mg/kg bw/d). The decrease in MCV values is not considered to be compound related since other red blood cell parameters, especially erythrocyte counts and hematocrit values were not affected. The increased leukocyte values were within the physiological range of rats. Therefore, the compound related effects are not relevant. No other effects, mostely related to immune response, were not affected. Histopathological examinations did not reveal any compound-related changes in the organs of the examined animals.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar rats – HanTac: WH

Sex:

male/female

Details on test animals or test system and environmental conditions:

Hylasco Biotechnology Pvt. Ltd.
Plot 4B, AKP,
Turkapally Village,
Shameerpet Mandal,
RR Dist, Telangana 500078
Rats were housed in an environment controlled room. The temperature maintained during the experiment was between 20 to 24°C and relative humidity was between 50 to 68%. The photoperiod was 12 hours light and 12 hours dark cycle. Adequate fresh air supply of 12.4 to 13.1 air changes/hour was maintained in the experimental room. The maximum and minimum temperature in the experimental room was recorded once daily. The relative humidity in the experimental room was calculated daily from dry and wet bulb temperature recordings.

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

Males: The vehicle and dose formulation were administered orally by gavage at approximately the same time each day (varying by ± 3 hours), for a period of ten weeks during pre-mating and a two-week mating period. Further, P (F0) males were dosed in the same manner until termination at the time when F1 animals were weaned after which they were sacrificed.
Females: The vehicle and dose formulation were administered orally by gavage at approximately the same time each day (varying by ± 3 hours), ten weeks prior to the mating period and continued through mating, pregnancy and up to weaning of their litters. The F1 offsprings received further administration with the vehicle and dose formulations from weaning to adulthood.
The dose volume administered to each rat was at an equivolume of 10 mL/kg body weight throughout the study. The dose volume was adjusted based on the most recent body weight and adjusted according to the body weight recorded at different intervals.
Following is the schematic representation of treatment schedule for various cohorts. Necropsy

Parents (P) Vehicle and dose formulation administration through oral gavage
Pre-mating Mating Post-mating
Males 10 Weeks 2 Weeks 6 Weeks
Females 10 Weeks 2 Weeks Pregnancy Lactation
F1 In-utero development Pre-weaning
Weaning & Selection for F1 generation

Necropsy

F1 Cohorts Vehicle and dose formulation administration through oral gavage Assessment
1A (20 Males and 20 Females/group) PND 107 Reproductive
1B (20 Males and 20 Females/group) PND 108 Reproductive

Details on mating procedure:

One female was placed with one male from the same group in a 1:1 ratio. Cohabitation was continued until there is evidence of sperms in the vaginal smear and /or vaginal plug or for a maximum period of 2 weeks. Subsequently, pregnant females were housed individually until lactation day 21. All the mated females were maintained for a maximum period of 25 days from the day of sperm positive. Females that had not littered were sacrificed after 25 days from the day they are found sperm positive (by vaginal smear examination).
The day of confirmed mating was designated as Gestation Day 0 (GD 0). The pre-coital time was calculated for each female.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For active ingredient (a.i.) concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 and 2nd (Day 42), 3rd (Day 77) and 6th (Day 181) months of the treatment period and analysed in-house. For each set, composite samples was drawn in three replications and in case of control, composite sample in one replication was drawn.
The analysis was done as per the method validated under Eurofins Advinus Study No.: G17947. One set of samples were analyzed for concentration analysis and other set (second set) of samples were stored at ambient conditions for reanalysis purpose as a backup and the second set of samples were discarded, when the analysis results of first set of samples were within the acceptable limits.
The samples of Day 1 and Day 181 were within the acceptable specification limits.
During month 2 (Day 42, 28 May 2019), the analysis results of G2 and G3 were out of specification in both set of samples. This could be due to sampling or processing error. Hence, the samples for G2 and G3 were collected from the formulations prepared on 29 May 2019 to confirm the concentration. The analysed results were within the specified limit as per study plan.
During month 3 (Day 77, 02 July 2019), the analysis results first set of samples were out of specification for G3 and G4. Hence, the backup samples were anlysed on 03 July and the results of G3 were within specified limit, while G4 were not specified limit as per study plan. Hence, the samples collected from the formulations prepared on 04 July were sent for analysis (G4) and the results were within the specified limit.
On both these occasions, the error could happen while during sampling or processing. Throughout the study period, the same procedure for the formulation preparation was followed. The formulations were freshly prepared on daily basis prior to administration. Further, the degree of the deviation of resultsthat failed to be within the given specification limits was rather low when compared to the dose spacings. . Hence, this will not affect the integrity of the study results.
Overall, formulations were considered acceptable as the mean results were within ± 20 % of the theoretical concentration and the relative standard deviation (RSD) was less than 20 %

Duration of treatment / exposure:

Start: 17 April 2019
End: 13 November 2019

Frequency of treatment:

Daily

Details on study schedule:

Study initiation date: 26 March 2019
Experiment start date: 29 March 2019
Acclimatization: Start: 29 March 2019, End: 02 April 2019
Pre-treatment period: Start: 03 April 2019, End: 16 April 2019
Treatment start: Start: 17 April 2019, End: 13 November 2019
Experiment completion date: 16 December 2019
Submission of draft report: 23 December 2019
Study completion date: 31 July 2020

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

25

Control animals:

yes

yes, concurrent no treatment

yes, concurrent vehicle

yes, plain diet

Details on study design:

GENERATION: F0
Group
No. Group Colour of
cage card Dose Concen-tration Dose volume No. of rats Sex Rat Numbers
(mg/kg Bwt/day) (mg/mL) (mL/ kg Bwt/day) From To
G1Vehicle Control White 0 0 10 25 M Rx591 Rx615
25 F Rx616 Rx640
G2 Low dose Yellow 100 10 10 25 M Rx641 Rx665
25 F Rx666 Rx690
G3 Mid dose Green 300 30 10 25 M Rx691 Rx715
25 F Rx716 Rx740
G4 High dose Pink 1000 100 10 25 M Rx741 Rx765
25 F Rx766 Rx790

GENERATION: F1
Group
No. Group Colour of
cage card Dose Concen-tration Dose volume No. of rats Sex Rat Numbers
(mg/kg Bwt/day) (mg/mL) (mL/ kg Bwt/day) From To
Cohort 1A: Reproductive Parameters Assessment and Splenic lymphocyte subpopulation analysis
G1A Vehicle ControlWhite 0 0 10 20 M Rx791 Rx810
20 F Rx811 Rx830
G2A Low dose Yellow 100 10 10 20 M Rx831 Rx850
20 F Rx851 Rx870
G3A Mid dose Green 300 30 10 20 M Rx871 Rx890
20 F Rx891 Rx910
G4A High dose Pink 1000 100 10 20 M Rx911 Rx930
20 F Rx931 Rx950
Cohort 1B: Reproductive Parameters Assessment
G1B Vehicle ControlWhite 0 0 10 20 M Rx951 Rx970
20 F Rx971 Rx990
G2B Low dose Yellow 100 10 10 20 M Rx991 Rx1010
20 F Rx1011 Rx1030
G3B Mid dose Green 300 30 10 20 M Rx1031 Rx1050
20 F Rx1051 Rx1070
G4B High dose Pink 1000 100 10 20 M Rx1071 Rx1090
20 F Rx1091 Rx1110

Parental animals: Observations and examinations:

9.1. General Clinical signs and Mortality
All rats were observed for morbidity and mortalities twice daily i.e., once in the morning and once in the afternoon except during holidays wherein the observation was done only once during morning hours.
All rats were observed for clinical signs twice daily (pre and post dose) during the treatment period. On the days of scheduled detailed clinical examination (once weekly as described in 9..2), clinical signs (after dosing) was included as a part of detailed clinical observations except on Day 1 wherein detailed clinical examination was done prior to the treatment and observations for general clinical signs was done after dosing the animals.
9.2. Detailed Clinical Examination
Detailed clinical examination was done prior to treatment on Day 1 and at weekly intervals thereafter (±2 days) during treatment period. On the days of detailed clinical examination, observation for general clinical signs was not performed except on Day 1.
During detailed clinical examination, all rats were observed for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards).
9.3. Body Weight
i) Individual body weights of males were recorded initially and at weekly (±1 day) intervals thereafter. Individual body weights of females were recorded initially and at weekly (±1 day) intervals thereafter till the confirmation of mating with males. Body weight of F1 generation animals was weighed once every two days during first two weeks after weaning and weekly intervals thereafter.
ii) All dams were weighed on Gestation Days (GD) 0, 2, 4, 7, 9, 11, 4, 16, 18 and 20 and on lactation days 0, 4, 7, 14 and 21.
9.4. Food Consumption
Cage wise food consumption was calculated by using the food consumed at weekly interval per cage and dividing by the number of rats per cage and the number of days in the intervening period to determine the food intake/rat/day.
Food consumption was not measured during the cohabitation period.
Food consumption of pregnant dams was recorded on GD 7, 14 and 20 and on Days 4, 7, 14 and 21 of lactation period.

Oestrous cyclicity (parental animals):

F0 Females: Vaginal smear was examined and the stage of oestrous cycle was recorded daily for two weeks before start of the treatment to select the females with regular 4-5 days cyclicity for the study. Vaginal smears was also monitored daily three weeks prior to mating to evaluate the regularity of oestrous cycle. Vaginal smears were also examined daily during the mating period to determine the Day ‘0’ of pregnancy and calculate the pre-coital time. The time interval (in days) from the diestrus of an oestrous cycle to the next diestrus was considered as the oestrous cycle length of an animal.
F1 Females in Cohort 1A: Vaginal smear was examined and the stage of oestrous cycle was recorded daily from onset of vaginal patency to first appearance of cornified smears in order to determine the time interval between these two events. The vaginal smears were also examined and the stage of oestrous cycle was recorded daily to evaluate the regularity of estrous cycle for all F1 females in cohort 1A for a period of two weeks commencing PND 79.
For adult F0 and F1 females, a vaginal smear was examined on the day of necropsy to determine the stage of the oestrous cycle.

Sperm parameters (parental animals):

Sperm parameters were measured in all F0 generation and all cohort 1A males.
Sperm motility was evaluated using the sperm samples collected from the right vas deferens, immediately after necropsy using Hamilton-Thorne TOX-IVOS (version 12) sperm analyzer. The vas deferens was excise approximately one cm near the cauda epididymis in the shortest time possible after anaesthetising the animal. The vas deferens was cut into 2 to 3 pieces and collected in 2 mL of the medium in a labelled centrifuge tube and kept in a water bath at the temperature range of 34ºC to 37ºC. The stage temperature was maintained between 36.6ºC and 37.4ºC in the acquire screen. An aliquot of 10µL of sample after mixing was loaded it into the 2X-CEL chamber slides with cover glass. The five fields were selected for analysis from the acquire screen.
For morphological evaluation of sperms, smears were made using sperm samples collected from right vas deferens immediately after necropsy, fixed with acetone for evaluation.
At termination, testis and epididymis weights were recorded for all F0 and F1 (cohort 1A) males.
The right testis and right epididymis were collected and frozen for enumeration of homogenization detergent resistant testicular spermatids and cauda epididymal sperm reserves, respectively

Litter observations:

a. At birth (lactation day 0), all the pups (both dead and alive) in a litter from each dam were observed for number and sex of pups, stillbirths, live births, and the presence of gross anomalies (externally visible abnormalities, including cleft palate; subcutaneous haemorrhages; abnormal skin colour or texture; presence of umbilical cord; lack of milk in stomach; presence of dried secretions). In addition, the first clinical examination of the neonates was also included a qualitative assessment of body temperature, state of activity and reaction to handling.
b. The number of pups born (litter size), sex and individual pup body weight of male and female pups on days 0 and 4 were recorded.
c. On day 4 after birth, the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, five pups per sex per litter. Blood samples were collected from two of the surplus pups of either sex, pooled, and used for determination of serum T4 and TSH levels. Whenever the number of male or female pups prevents having five of each sex per litter, partial adjustment (for example, six males and four females) was done. No pups were eliminated when litter size dropped below the culling target (10 pups/litter). In case of availability of only one pup above the culling target, only one pup was eliminated and used for blood collection for serum T4 assessments.
d. After standardization, the individual pup body weight was recorded on day 7, 14 and 21 of lactation.
e. The ano-genital distance (AGD) of each pup was measured on PND 0 and pup body weight was recorded. Ano-genital distance ratio was calculated by dividing the ano-genital distance from cube root of body weight.
f. The number of nipples/areolae in male pups were counted on PND 13.
g. All the dead and sacrificed pups were examined for malformations and subjected to gross pathological examination.
h. The litters were observed daily in order to note the number of alive, dead and cannibalized pups.
i. In addition to daily clinical observations, any abnormal behavior of the offspring were recorded.
j. Fertility index for dams, sires as well as the pup survival index until lactation day 21 was calculated.
k. On the day of weaning (Lactation day 21), the following number of pups were randomly selected to different cohorts for post weaning evaluations.


Parental (P) generation Cohort Designation No. of animals/cohort Sexual Maturation Approximate age at necropsy (weeks)
Target is 20 litters/group 1A Reproductive 20M+20F Yes 14 -15
1B Reproductive 20M+20F Yes 14 - 15

l. All selected F1 females were observed for vaginal opening commencing from PND 27 and males for balano-preputial separation commencing from PND 37. The body weights were also recorded for pups selected for F1 generation at the time of vaginal patency or balano-preputial separation.

Postmortem examinations (parental animals):

Parental males: After completion of mating, the parental males were sacrificed in parallel to females at weaning.
Parental females: Parental females were sacrificed on PND 22 along with offspring not selected for various cohorts. The uteri of all F0 females were examined for the presence and number of implantation sites, in a manner which does not compromise histopathological evaluation. For apparently non-pregnant rats, the uteri was stained with ammonium sulphide solution to identify the pre-implantation loss of the embryos.
For F1 animals:
Cohort 1 1A PND 99-107 (Approximately 14-15 weeks)
1B PND 100-108 (Approximately 14-15 weeks)
All adult animals and pups including found dead were examined macroscopically for any structural abnormalities and pathological changes. Special attention was paid to the organs of the reproductive system. The rats were subjected to detailed necropsy and findings recorded.
The animals sacrificed at term were [10 randomly selected males and females of F0 and Cohort 1A were fasted overnight (water allowed)] weighed and exsanguinated under isoflurane anaesthesia.
Dead pups were examined for possible defects and/or cause of death. Dead pups were persevered in 10% NBF and discarded before finalization of report

All Parental animals and F1 Cohort 1A animals:
On completion of the gross pathology examination, the tissues/organs noted in following table were collected and weighed from all rats. Terminal fasting body weights were recorded for all animals immediately prior to terminal sacrifice and used in calculation of relative organ weights. The organ weight ratios (organ to body weight and brain weight) as percentage of fasting body weight were determined and presented in the report. Paired organs were weighed together and combined weight was presented. The tissues were preserved in 10 % Neutral Buffered Formalin (NBF) unless noted otherwise:
F1 Cohort 1B animals:
On completion of the gross pathology examination, the tissues/organs noted in following table were collected and weighed. The organ weight ratios as percentage of fasting body weight was determined and presented in the report. Paired organs were weighed together and combined weight was presented. The tissues were preserved in 10 % Neutral Buffered Formalin (NBF) unless noted otherwise.

Postmortem examinations (offspring):

F1 weanlings not selected to form cohorts:
The pups not selected for cohorts were terminated after weaning, on PND 22. On completion of the gross pathology examination, the tissues/organs noted in following table were collected and weighed from all available litters. The tissues were preserved in 10 % Neutral Buffered Formalin (NBF).
Sl. No. Organ/Tissue Organ
Weight Collection & Preservation
1 Brain X X
2 Mammary gland X
3 Spleen X X
4 Thymus X X
X: Organs weighed, collected and preserved.

Statistics:

Data captured using ProvantisTM: Parameters of body weight, oestrous cycle, ano-genital distance, organ weights, terminal fasting body weight, laboratory Investigations - Haematology, Coagulation, Clinical Chemistry and Urinalysis parameters were analyzed using built-in statistical tests.
Derived data like body weight change, food consumption, ano-genital ratio, oestrous cycle length, organ weight ratios, post implantation loss (%), number of nipples/areolae in male pups, no. of implantations, pre-coital interval, mean litter size, sex ratio and gestation length (days), mating, fertility and survival indices were also analyzed using above mentioned methods.
Hormone data was analysed using the validated package in Excel and also using licensed copies of SYSTAT Statistical package ver.12.0. All quantitative variables were tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene’s test) within the group before performing a one-factor ANOVA modeling by treatment groups. Non-optimal (non-normal or heteroschedastic) data was transformed, before ANOVA is performed. Comparison of means between treatment groups and control group was done using Dunnett’s test when the overall treatment, ‘F’ test was found to be significant. For two groups, the comparisons of mean between treatment and control group was done using student‘t’ test.
All analyses and comparisons were evaluated at the 5% (p<0.05) level. Statistically significant differences (p<0.05), indicated by the aforementioned tests were designated throughout the report as stated below:
*: Significantly different from the vehicle control group

Reproductive indices:

Reproductive Performance Data of Parents
a. Male mating index (%)

No. of males with confirmed mating
= ----------------------------------------------------- x 100
No. of males cohabited

b. Female mating index

No. of females with confirmed mating
= ----------------------------------------------------- x 100
No. of females cohabited

c. Male fertility index (%)

No. of males impregnating a female
(Confirmed at necropsy)
= ----------------------------------------------------- x 100
No. of males cohabited

d. Female fertility index (%)

No. of pregnant females (confirmed at necropsy)
= --------------------------------------------------------------- x 100
No. of females cohabitated

e. Mean no. of implantations

Total no. of implantations
= --------------------------------------------------------
No. of dams used for implantation count

f. Post implantation loss (%)

No. of implantations - No. of live fetuses
= ------------------------------------------------------- x 100
No. of implantations

Offspring viability indices:

Data on Litters
a. Mean litter size

Total no of pups on day 0
= ---------------------------------------
No. of females littered

b. Mean viable litter size

No. of viable pups on day 0
= -----------------------------------------
No. of females littered

c. Live birth index (%)

No. of viable pups born (at first observation)
= -------------------------------------------------------------- x 100
Total no. of pups born (at first observation)

d. Day 4 survival index (%)

No. of viable pups on lactation day 4
= ----------------------------------------------------- x 100
No. of viable pups born

e Day 7 survival index (%)

No. of viable pups on lactation day 7
= ---------------------------------------------------------------- x 100
No. of viable pups retained on lactation day 4

f. Day 14 survival index (%)

No. of viable pups on lactation day 14
= --------------------------------------------------------------- x 100
No. of viable pups retained on lactation day 4

g. Day 21 survival index (%)

No. of viable pups on lactation day 21
= ---------------------------------------------------------------- x 100
No. of viable pups retained on lactation day 4

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:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

The oral administration of EXOLIT OP 1230 in F0 parents and Cohorts 1A and 1B in Wistar rats had no effects on general health, body weights, food consumption. There were no significant exposure-related effects on organ weights in F0 and F1 adults. The test item had no effects on estrous cyclicity (P1, 1A and 1B) or P reproductive indices, including mating, fertility, time to mating, gestation length, post-implantation loss. Litter sizes, pup survival, sperm parameters, and ovarian follicle counts were unaffected. There were no exposure-related histopathological changes in reproductive organs. Overall, there was no indication of systemic or reproductive toxicity by EXOLIT OP 1230 at any of the doses tested.
Endocrine toxicity includes an evaluation of androgen, estrogen, and thyroid endpoints, as well as other glands that contribute to the maintenance of homeostasis.
Androgen dependent endpoints:
In P generation males, statistically significant decrease in testis parenchyma weight along with decrease in detergent and homogenization resistant testicular spermatid counts (number of spermatid per testis and number of spermatid per gram of parenchyma) were observed at 1000 mg/kg/day. The decreases were mainly associated with the small sized testes in few rats at 1000 mg/kg/day and the testis parenchyma weight as well as the testicular spermatid count values were within in-house historical control data of two generation reproduction toxicity study in Wistar rats. The observed decrease of relative testes value of F0G3 group in males of the P generation, although statistically significant, was well within the in-house historical control data range of two generation reproduction toxicity study in Wistar rats. However, there were no significant changes in the epidydimis weight (total and cauda) and the epididymal sperm counts. The sperm motility (total and progressive motility) and the sperm morphology (normal and abnormal sperms) were also within the normal biological variation and there were no significant findings in the weight and morphology of accessary sex glands. In addition, no treatment-related changes were observed in the fertility indices of sires and dams. In the absence of all the above changes, the decrease in testicular weight and spermatid count at 1000 mg/kg/day were not related to test item administration.

Androgen-dependent endpoints are summarized below: Report attached.

Sperm parameters: Report attached

Organ weights: Report attached

The motility, morphology, spermatid/sperm counts, organ weights/ratios, gross and histopathological changes were not affected by test item administration in F1 generation males (Cohort 1A).
Based on this data, the androgenicity or anti-androgenicity end points in the treated groups were comparable to vehicle control.

Estrogen-sensitive endpoints: Report attached

Thyroid dependent endpoints and other glands:
Thyroid assessment included thyroid hormones (T4, and TSH), thyroid weights, and/or histopathology. There were no significant changes in the thyroid assessment parameters.
There was no indication of alterations in adrenal function as there were no effects on absolute or relative adrenal gland weights and no exposure-related histopathological findings at any of EXOLIT OP 1230 exposure level.
Decision not to breed a second generation:
Based on the results of this Extended One Generation Reproduction Toxicity study, there were no indications to breeding a second generation.
Conclusion:
Based on the above mentioned results, oral administration of EXOLIT OP 1230 did not result in pre- and postnatal systemic toxicity neither in pregnant and lactating females, nor in young and adult offspring. There were no effects on key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood. The test item had no effect on the integrity and performance of the adult male and female reproductive systems such as gonadal function, oestrous cycle, epididymal sperm maturation, mating behaviour, conception, pregnancy, parturition, and lactation.
Overall, there was no indication of systemic or reproductive toxicity by EXOLIT OP 1230 at any of the dose levels tested.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effect up to the highest dose

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Developmental immunotoxicity:

no effects observed

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effect up to the highest dose

Key result

Reproductive effects observed:

no

Conclusions:

The registration substance was investigated for the reproduction toxicity according to the OECD Guideline 443. No effect was found up to the highest dose of 1000 mg/kg/day. No indication of fertility toxicity was found.

Executive summary:

The registration substance was investigated for the reproduction toxicity according to the OECD Guideline 443.

Study Design

The test item was suspended in 0.5% Sodium carboxy methyl cellulose with 0.1% Tween 80 in Milli-Q®water (vehicle) and administered to sexually-mature parental rats at the graduated dose levels of 100, 300 and 1000 mg/kg/day for low dose (G2), mid dose (G3) and high dose (G4) group rats, respectively. The rats in the vehicle control group (G1) received vehicle alone. The dose volume administered was 10 mL/kg body weight. Each parental group consisted of 25 male and 25 female rats.

The parental (F0) generation rats were administered for ten weeks during pre-mating and a two-week mating period. Parental (F0) males were further administered until weaning of the F1offsprings. They were administered for a period of total 16 weeks. Dosing of the parental females were continued during pregnancy and lactation until termination after the weaning of their litters (i.e.16 -17 weeks of treatment). On Lactation Day 21, pups were selected to form 1A and 1B of F1Cohorts for post-weaning evaluations. Cohorts 1A and 1B were comprised of 20 males and 20 females. F1 offspring received further treatment with the test item from weaning until terminal sacrifice. Animals in the 1A and 1B Cohorts were used for evaluation of reproductive function.

Observation Parameters:

The F0 generation rats were observed for clinical signs once daily. Body weight and food consumption were recorded at weekly intervals in either gender, prior to mating and during post-mating in males. Female rats were also weighed on Gestation Day (GD) 0, 2, 4, 7, 9, 14, 16, 18 and 20 and on Lactation Day (LD) 0, 4, 7, 14 and 21. Food consumption was measured during GDs 0-7, 7-14 and 14-20 and LDs 0-4, 4-7, 7-14 and 14-21. The number, weight, survival and mortality of pups were recorded during the lactation period. The ano-genital distance of each pup was measured on LD 0. All the survived male pups were examined for the appearance of nipples/areolae on post-natal day 13. The blood samples were collected from available two pups/litter in F1litter on LD 4 and 22 for thyroid hormone analysis.

The blood samples were collected for clinical pathology and thyroid hormone analysis from 10 randomly selected F0 males and females, prior to necropsy. All F0 males were sacrificed after mating in parallel to females at weaning and female rats were sacrificed at weaning. The number of implantation sites were recorded. All F0 male and female rats were subjected to detailed necropsy at sacrifice and study plan specified organs were collected. Andrological assessment like sperm motility, morphology, testicular spermatid and cauda epididymal sperm counts were evaluated from all F0 and F1 cohort 1A males.

Clinical signs, body weights and food consumption were recorded for the pups selected in F1 generation (20 pups/group from each gender and Cohorts) that were randomly chosen for continued evaluation for different Cohorts. All rats were evaluated for sexual maturation(vaginal opening and preputial separation).Vaginal smear was examined and the stage of oestrous cycle was recorded daily from onset of vaginal patency to first appearance of cornified smears to determine the time interval between these two events. Randomly selected 10 males and females in cohort 1A weresubjected for splenic lymphocyte subpopulation analysis (CD4+ and CD8+ T lymphocytes, B lymphocytes, and natural killer cells) by flow cytometer using one half of the spleen collected during necropsy. Animals of Cohort 1B were subjected to detailed necropsy and protocol specified organs were weighed and preserved.

Blood samples were collected for clinical pathology and thyroid hormone analysis from 10 randomly selected males and females of 1A Cohort, prior to necropsy. All rats of F1 Cohorts were subjected to detailed necropsy at sacrifice and organs were collected. Andrological assessment like sperm motility, morphology, testicular spermatid and cauda epididymal sperm counts were evaluated for Cohort 1A males.

Histopathological examination was carried out on the organs / tissues of F0 and F1 Cohort 1A animals.

Results for F0 Generation/F1 Pre-weaning:

Clinical signs & Mortality: No clinical signs and mortality were observed during clinical examination and daily observations.

Body Weights: The mean body weights and body weight gains were unaffected throughout the treatment period in males and ten weeks pre-mating period in females at all the tested doses when compared to vehicle control group.

Food consumption: The food consumption was unaffected by the treatment at all the tested doses in either sex.

Maternal body weights and food consumption: The maternal body weight and food consumption measured during different interval of gestation and lactation period was unaffected by the treatment at all the tested doses.

Fertility parameters: Treatment had no effect on pre-coital time, gestation length, oestrous cycle length, mating and fertility parameters in both sexes.

F1Litters: There were no treatment-related effects on the uterine/implantation data, mean litter size and mean viable litter size. There were no external abnormalities in live or dead pups in any of the groups. Litter mean pup body weight of F0G3 group was significantly reduced on lactation days 4 and 7, compared to corresponding control animals. Despite initial lower body weights of F0G3 pups, body weight development was not influenced and reached comparable levels during the lactation phase in all groups. No treatment-related changes in the ano-genital distance and ano-genital ratio were observed at any of the doses tested when compared to the vehicle control group. The male pups did not exhibit areola/nipple retention at any of the doses tested.

Pathology: There were no test item-related changes observed in clinical pathology (haematology, coagulation, clinical chemistry and urinalysis), thyroid hormones profile(total T4 and TSH), sperm parameters (motility, morphology, homogenization detergent resistant testicular spermatids and cauda epididymal sperm reserves), terminal fasting body weights, organ weights/their ratios in both parental male and female rats. There were no gross and microscopic changes observed in both sexes.

Results for F1 Generation Post-weaning Evaluation in Cohorts 1A and 1B:

Clinical Signs & Mortality: No clinical signs and mortality were observed during clinical examination and daily observations.

Body Weights: The mean body weights and total body weight gains were unaffected throughout post-weaning treatment period at all the tested doses in both sexes.

Sexual Maturation: The acquisition of the sexual maturation indicator such as vaginal opening and balano-preputial separation occurred in different cohorts were comparable to vehicle control group indicative of normal sexual maturation of animals.

Oestrous Cycle Evaluations in Cohort 1A animals: The average days required for first appearance of cornified cells in the vaginal smear after the occurrence of vaginal patency was unaffected by the treatment. The oestrous cyclicity was evaluated for a period of two weeks commencing around PND 79. The mean oestrous cycle lengths in the treated groups were comparable to vehicle control group.

Splenic Lymphocyte Population Analysis in Cohort 1A: The splenic lymphocyte subpopulation (CD4+ and CD8+ T lymphocytes, B lymphocytes, and natural killer) was analysed using flow cytometry from randomly selected ten male and ten female rats. The results indicated that the lymphocyte subpopulation was unaffected by the treatment.

Pathology: There were no test item-related changes with respect to haematology, coagulation, clinical chemistry, urinalysis parameters, thyroid hormones profile, sperm parameters, terminal fasting body weights, organ weights/their ratios, gross and histopathology in both male and female rats at all the dose levels tested in Cohort 1A animals. There were no test item-related changes observed in terminal fasting body weight, organ weight/their ratios and gross changes in both the sexes of Cohort 1B animals.

 

Conclusion:

Based of the obtained results, oral administration of EXOLIT OP 1230 did not result in pre- and postnatal systemic toxicity neither in pregnant and lactating females, nor in young and adult offspring. There were no effects on key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood. The test item had no effect on the integrity and performance of the adult male and female reproductive systems such as gonadal function, oestrous cycle, epididymal sperm maturation, mating behaviour, conception, pregnancy, parturition, and lactation.

Overall, there was no indication of systemic or reproductive toxicity by EXOLIT OP 1230 at any of the dose levels tested. NOAEL of 1000 mg/kg/d was obtained.

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:

one valid reproduction toxicity screening test (OECD 421) and one valid EOGRT (OECD 443)

Effects on developmental toxicity

Description of key information

The registration substance did not induce any toxicity in the developmental toxicity study in rats up to the highest dose of 1000 mg/kg/d.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 August 2012 - 18 June 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

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
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: approx. 12-13 weeks old
- Weight at study initiation: males: 311 - 350 g - (mean: 329 g, ± 20% = 263.20 – 394.80 g); females: 200 - 239 g - (mean: 217 g, ± 20% = 173.42 – 260.12 g)
- Fasting period before study: none
- Housing: - Full barrier in an air-conditioned room
- Diet (e.g. ad libitum): Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 0856)
- Water (e.g. ad libitum): Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 3°C
- Humidity (%): 55 +- 10%
- Air changes (per hr): 10 x / hour
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

IN-LIFE DATES: From: 28 August 2012 To: 29 November 2012

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

Polyethylenglycol 400

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

The test item was weighed into a tarred plastic vial on a precision balance.
The test item was suspended in Polyethylenglycol 400.
The test item formulation was prepared freshly on each administration day before the administration procedure. The time of preparation and time of dosing was recorded for all dosing formulations.
Homogeneity of the test item in the vehicle was maintained by vortexing the prepared suspension thoroughly before every dose administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle was selected as suggested by the sponsor based on the test item’s characteristics and testing guideline
- Concentration in vehicle: 20, 60, 200 mg/ml
- Amount of vehicle (if gavage): 5 ml / kg BW
- Lot/batch no. (if required): 1101401034
- Purity: 100 %

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The assessment of homogeneity as well as a determination of the nominal concentration of the test item in the vehicle was performed at various intervals.
Samples for analysis of the dose formulations of the test item in the vehicle (nominal concentration) were taken in the first and last week of the study for all doses (8 samples in total).
Samples for homogeneity were taken from the top, middle and bottom of the high dose and low dose preparation. Samples were taken in the first and last week of the study (18 samples in total).
Samples for stability analysis were taken in the first week of the study, 0 hours after the preparation and another sample 6 hours after the preparation (at room temperature), from high, medium, and low dose formulations (6 samples).
All formulation samples were stored at -20° C and were analysed after completion of the in-life phase of the toxicity study at BSL BIOSERVICE Scientific Laboratories GmbH. The procedures followed for the sample analysis were mentioned in the phase plan (BSL phase study no. 124188).

Details on mating procedure:

- Impregnation procedure: [cohoused]
- If cohoused: Mating was performed using a ratio of 1:2 (male to female). Females were paired for cohabitation in batches in order to regularise the number of animals for terminal sacrifice on a particular day. The subsequent morning and the next morning onwards, the vaginal smear of each female was checked to confirm the pregnancy. The day on which sperms were observed in the vaginal smear was considered as gestation day ‘0’. Mated females were assigned in an unbiased manner to the control and treatment groups ensuring that the mean body weights were comparable to each other. Each animal was assigned a unique identification number
- M/F ratio per cage: 1:2
- Length of cohabitation: maximum of 14 days
- Further matings after two unsuccessful attempts: N.A.
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: [sperm in vaginal smear] referred to as [day 0] of pregnancy
- Any other deviations from standard protocol: None

Duration of treatment / exposure:

The test item is orally administered daily in graduated doses to several groups of pregnant females from the gestation day (GD) 5 to gestation day (GD) 19

Frequency of treatment:

Once per day. 7 days per week

Duration of test:

On GD 20, i.e. the day prior to the expected day of delivery, the presumed pregnant females are subjected to a caesarean section.

No. of animals per sex per dose:

The 4 groups comprised 25 female Wistar rats, except the HD group where only 24 animals were integrated into this study. This was due to the total amount of pregnant females.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response and a NOAEL.
- Rationale for animal assignment (if not random): Mated females were assigned in an unbiased manner to the control and treatment groups ensuring that the mean body weights were comparable to each other

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing


DETAILED CLINICAL OBSERVATIONS: No


BODY WEIGHT: Yes
- Time schedule for examinations: The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment. The sperm positive females were weighed during gestations days 0, 5, 8, 11, 14, 17 and 20. Males were not weighed in this study except on the day of their arrival.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: At the time of termination, the dam (presumedly pregnant female) was examined macroscopically for any structural abnormalities or pathological changes which may have influenced the pregnancy.
Immediately after the termination or as soon as possible after death, the uteri were removed and the pregnancy status of the dams was confirmed. Uteri that appear non-gravid were further examined by staining with 10 % ammonium sulphide solution to confirm the non-pregnant status.
Each gravid uterus with the cervix was weighed.
The number of corpora lutea was counted for pregnant animals. The uterine contents were examined for embryonic or foetal deaths as well as the number of viable foetuses. The degree of resorption (late and early) was confirmed in order to help estimate the relative time of death of the conceptus. The position and number of foetuses in each uterine horn was also recorded.

Ovaries and uterine content:

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

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Statistics:

A statistical assessment of the results of the body weight, food consumption was performed by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Foetal evaluation parameters like external, visceral, craniofacial and skeletal parameters were analysed using a Chi-square test. The statistics were performed with GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: clinical symtoms

Details on maternal toxic effects:
No mortalities were recorded in the C group or any treatment group.
Clinical symptoms were found in all groups (C and treatment groups) and some of them could be found increased in MD and HD group. The most prominent were diarrhea (9/24 HD animals, 4/25 C animals), diarrhea directly after administration (8/24 HD animals, 2/25 C animals), moving the bedding immediately after administration (20/24 HD animals, 8/25 MD animals, 0/25 C animals), slight piloerection (6/24 HD animals, 4/25 MD animals, 0/25 C animals), slight piloerection immediately after administration (8/24 HD animals, 5/25 MD animals, 0/25 C animals), slight salivation immediately after administration (4/24 HD animals, 2/25 MD animals, 1/25 C animals). These symptoms could be concerned with a toxicological relevance in HD group (mostly due to diarrhoea) and with general discomfort of the animals in MD and HD groups. However, since no influence on weight development was observed a clear toxicological relevance cannot be assumed.
Throughout the treatment period, body weights and body weight gain were within the normal range of variation for this strain and no difference could be found between C and treatment groups.
In correlation to the body weight and body weight change, the food consumption increased with the progress of the study in all groups. In treatment groups there was a slightly increased food consumption which was not dose-dependent over all groups.
The statistical analysis of prenatal data revealed no significant effect on each of the parameters. An increased percentage of pre-implantation loss in the LD group is assumed to be incidental, since neither MD nor HD group were affected. An increased percentage of post-implantation loss in the HD group is not assumed to be test item related due to the missing statistical significance combined with the high standard deviation
No treatment related change was observed during the macroscopic observation. Only incidental findings were recognized.

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: no significant effect up to the highest dose level

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No statistical significant effect was found in treatment groups of litter data such as mean litter weight, number of males, number of females, total number of pubs, total litter weight, male litter weight, and female litter weight. No influence of the test item on any of the parameters was observed.
Few gross external abnormalities were seen among the control and treatment groups but none of them was statistical significant and all of them are assumed to be incidental.
Skeletal examination of the Alizarin red stained foetuses revealed a range of abnormalities which were of a type or which occurred at an incidence comparably lower in treated groups when compared to the control group.
Several abnormalties were increased when compared to C group which were either statistically significant or in a range that it could be of toxicological relevance. These are as follows:
A 14th rudimentary rib (right side; p<0.05 C vs HD)) was found in 13 fetuses of HD group and in 5 fetuses of C group. The incidences were 3.73 % (C) and 12.26 % (HD). The number of litters affected were 4 (C) and 8 (HD). This could be test item related but cannot be assumed as malformation but as variation. Hence, a toxicological relevance is not assumed.
Increased fused frontal and parietal bones were recognized at the skull in treatment groups and the incidences of C vs LD groups were significantly different (p<0.05). In particular 2 incidences were counted in C group as well as 7 (LD), 6 (MD), and 6 (HD) incidences which were counted in treatment group. The percentage incidence of abnormalties was 1.49 % (C), 6.31 % (LD), 5.77 % (MD), and 5.66 % (HD). The number of litters with at least 1 incidence was 2 in C, LD, MD, and HD groups, respectively. Since there was no dose-dependency in percentage of incidences and since the same number of litters was affected, this finding is not assumed to be treatment related but incidental.
 
An incomplete ossification of the interparietal (skull) was significantly increased (p<0.01) in HD group when compared to C group. A percentage of 61.32 % was calculated as abnormal in HD group when compared to 44.03 % in C group. Furthermore, 16 (HD) and 20 (C) litters were affected. Due to the decrease in affected litters, this is not assumed to be test item related.
An incomplete ossification was also mentioned for frontal (skull) in treatment groups when compared to C group (not statistical significant). 14.15 % affected fetuses (HD), 10.58 % (MD), 9.91 % (LD), and 7.46 % (C) were calculated. The number of affected litters was 7 (C), 5 (LD), 8(MD), and 9 (HD). A test item relation is not assumed. No statistical significance could be calculated and the number of affected litters did not follow a dose response and showed only slight and variable changes.
An increased incomplete ossification was mentioned for the parietal (skull) for HD group when compared to C group (p<0.01). 48.11 % (HD) and 30.6 % (C) affected fetuses were counted. The number of affected litters was 15 in both groups. Hence, a test item relation is not assumed but an incidental case.
Several other differences among treatment groups and C groups were statistically significant but are not assumed to be treatment related and/or of toxicological relevance (due to lack of dose dependency and/or due to a higher incidence in C group).
Internal examinations of the fetal viscera by the free-hand-microdissection technique revealed a range of visceral abnormalities in all groups including the control.
A slightly increased percentage of concerned fetuses could be found for hemorrhigoc adrenal glands (both sides). 10.2 % (HD), 7.45 % (MD), 7.77 % (LD), and 4.92% (C) were counted / calculated. The number of affeceted litters was 3 (C), 7 (LD), 5 (MD), and 8 (HD). A statistical significance was not found and a dose-dependency is not present. Hence, it is not assumed that these slight increases of hemorrhagic adrenals are of test item related.
A statistical significant increase could be found for hemorrhagic lungs when compared C with MD group. However, no dose dependency could be found and in HD group just 1 incidence could be counted. Hence, this is not assumed to be treatment related.
Convoluted ureters (both sides) were found clearly and statistically (LD; HD vs C p<0.001; MD vs C p<0.01) incrased in treatment groups when compared to C group. In particular: fetal incidences of 3.31. % (C), 18.45 % (LD), 17.65 % (MD), and 22.83 % (HD) were evaluated. The amount of litters with at least 1 incidence was 4 (C), 12 (LD and MD), and 14 (HD). Although not dose dependent, this is assumed to be test item related. However, since the convoluted ureter is assumed to be a variation and no malformation, this is not assumed to be of toxicological relevance.
An increased percentage of dilated ureters could be found on both sides (not statistical significant). In particular, 2.46 % (C), 6.80 % (LD), 5.32 % (MD), and 8.16 % of all respective fetuses were affected. Furthermore, the number of affected litters was 3 (C) and 5 (LD, MD, HD). This coincides with a slight increase in dilated renal pelvis where 15.22 % (HD) and 8.26 % (C) of all fetuses were affected. However, no statistical significance was calculated and this finding is mostly interpreted as variation. Hence, a toxicological relevance is not assumed.
An increased percentage of a thin ventricular wall was recored in HD group (5.1 % of all fetuses) when compared to C group (1.64 % of all fetuses). The numbers of affected litters were 2 (C), 3 (LD), 1 (MD), and 4 (HD). Since no statistical significance is calculated and since the number of affected litters did not follow a dose response this is not assumed to be test item related but incidental.
Craniofacial examination by razor blade serial sectioning technique revealed a range of visceral abnormalities in all groups including controls. The statisitical analysis of the data mostly revealed no statistical significant and biological relevant differences in the number of incidences in treated groups as compared to the control group. Therefore, these findings are not to be considered to be treatment related and spontaneous in nature. The observed significant changes are not considered to be test item related, since only MD and LD groups were concerned.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: fetotoxicity

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

The registration substance was investigated for its developmental toxicity in rats according to the OECD Guideline 414. NOAEL of 1000 mg/kg/day was obtained for maternal as well as for fetal toxicity. No indication of developmental toxicity was found in rats.

Executive summary:

The registration substance was investigated for its developmental toxicity in rats according to the OECD Guideline 414. The registration substance was suspended in PEG and given to pregnant rats per gavage at doses of 0, 100, 300 and 1000 mg/kg/day from GD 5 -19. On day GD20 cesarian section was performed. No significant maternal toxicity was found up to the highest dose and no significant fetal toxicity was found up to the highest dose. NOAEL of 1000 mg/kg/day was obtained for maternal as well as for fetal toxicity. No indication of developmental toxicity was found in rats.

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:

one valid developmental toxicity study (OECD 414)

Toxicity to reproduction: other studies

Additional information

no further data mandatory

Justification for classification or non-classification

The registration substance was investigated for the reproduction toxicity according to the OECD Guideline 443. No effect was found up to the highest dose of 1000 mg/kg/day. No classification is justified for the endpoint fertility.

The registration substance was investigated for the developmental toxicity in rats according to the OECD Guideline 414. No effect was found up to the highest dose of 1000 mg/kg/day. No classification is justified for the endpoint developmental toxicity.