Ethylene bis[3,3-bis(3-tert-butyl-4-hydroxyphenyl)butyrate]

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

To evaluate the test substance DTB-glycolester for its fertility property, data from a chronic toxicity and carcinogenicity study in rats are available, from which the reproduction phase is reported here. In principle it follows a test design of a one-generation study conducted with young adults obtained from mothers which had been treated with the test substance at dietary levels of 0, 0.16, 0.4 or 1% during 30 days before mating, during gestation and lactation. The feeding of DTB-glycolester at dietary levels of 0, 0.16, 0.4 or 1% (corresponding to 0, 70, 200 and 500 mg/kg bw/d considering a diet conversion factor (ppm to mg/kg bw/d) of 20) to rats resulted in slight growth depression and relatively low food intake in male parent rats of the mid and high dose group. The only change observed in the litters consisted of slight growth retardation of the pups in the top dose group during the final stage of the lactation period. The other data on fertility, lactation performance and survival did not reveal any abnormalities. The NOAEL (P, F1) was considered to be 1% (corresponding to 500 mg/kg bw/d; highest dose tested in the one generation study) in diet.

These findings are in accordance with results from an OECD 421 screening study, performed in rats at dietary levels of 0, 3750, 7500 and 11250 ppm (i.e. For females, test item intake over study period (pre-mating, post-coitum, lactation) corresponds to 501, 1013 and 1320 mg test item/kg body weight. For males, test item intake over study period (pre-mating, post-coitum) corresponds to 271, 513 and 809 mg test item/kg body weight respectively). The NOAEL for reproduction was considered to be at least 11250 ppm, corresponding to 809 mg/kg bw/day in males and 1320 mg/kg bw/day in females.

Justification for selection of Effect on fertility via oral route:
This reproductive toxicity study performed with Ethylene-bis[3,3-bis(3-tert.-butyl-4-hydroxyphenyl)butyrate] was selected as the most relevant available reproductive toxicity study due to its reliability and since it provides a sensitive NOAEL.

Link to relevant study records

Reference

Reference 1

Endpoint:

one-generation reproductive toxicity

Remarks:

based on generations indicated in Effect levels (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Aug - Dec 1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well performed and reported non-guideline study with scientific sound design and sufficient reporting.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Rats were fed diets containing the test item at different levels and mated. The litters were reared and observations were made on fertility of females, number of young born per litter, sex ratio, grossly visible abnormalities, mortality, body weights and resorption percentage.

GLP compliance:

no

Remarks:

performed before GLP guidelines

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: The Central Institute for the Breeding of Laboratory Animals TNO, Zeist, The Netherlands
- Age at study initiation: 11 weeks
- Weight at study initiation: (P) Males: 268 g; Females: 179 g
- Housing: in groups of five in screen-bottomed, stainless steel cages
- Diet (e.g. ad libitum): CIVO basal diet (containing 29.7% yellow maize, 36% whole wheat, 11% defatted soy-bean mael, 4% meat scraps, 7% fish meal, dried whey, brewer's yeat, grass meal, soy-bean oil, vitamin preparations, trace mineralized salt, steamed bone meal), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 2 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23-25°C
- Humidity (%): 50%
- lighting 12 h daily

3 weeks acclimatisation

IN-LIFE DATES: From: Aug. 1978 To: May 1981 (end of subsequent chronic toxicity study)

Route of administration:

oral: feed

Vehicle:

other: CIVO basal diet

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency):
The test diets were normally prepared in batches once every 3 to 4 weeks.
The test material was thoroughly mixed into CIVO basal diet by means of a mechanical blender at levels of 0 (control), 0.16, 0.4 or 1.0%. The diets were stored in an unheated room at ambient temperatur

Details on mating procedure:

After the pre-mating period (30 days) the rats were mated within their diet group. Each male was housed with two females in a cage for one week. At week 2 and 3 of the mating period each male rat was transferred to another "mating" cage within the same diet group. So, three different males were available for each dam. After a mating period of three weeks, the females were caged individually, until their litters had been weaned.

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

no data

Duration of treatment / exposure:

Males: 7 weeks (4 weeks pre-mating, 3 weeks mating)
Females: 13 weeks (4 weeks pre-mating, 3 weeks mating, 3 weeks gestation, 3 weeks lactation)

Frequency of treatment:

continuously

Details on study schedule:

- Age at mating of the mated animals in the study: 15 weeks

Remarks:

Doses / Concentrations:
0.16%
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
0.4%
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
1.0%
Basis:
nominal in diet

No. of animals per sex per dose:

15 males/dose
30 females/dose

Control animals:

yes, plain diet

Details on study design:

no data

Positive control:

No

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
In the pre-mating period of 30 days food intake was recorded at weekly interval.

Oestrous cyclicity (parental animals):

no data

Sperm parameters (parental animals):

no data

Litter observations:

Records were made of the number of pups in each litter, sex ratio at birth and the weight of the litter at day 1,4, 14 and 21 of lactation. Litters containing more than 8 siblings were randomly reduced to 8 on day 1 of lactation, in order to equalize the stress of lactation among the dams.

Postmortem examinations (parental animals):

After weaning their young, the mothers were sacrificed and the implantation sites in the uterus were counted after staining with ammonium sulfide solution. The males were discarded.

Postmortem examinations (offspring):

n.a., since the offspring was used a subsequent combined chronic toxcity/carcinogenicity study (please refer to study report no. R 6693; IUCLID Chapter 7.5.1; WoE_130 week oral toxicity (diet)_rat_TNO_1982)

Statistics:

All data were evaluated by means of the Student t-test.

Reproductive indices:

- female fertility index = (number of pregnant females/number of females placed with males) x 100
- gestation index = (number of females with live pups/number of females pregnant) x 100
- post-implantation loss = [(number of implantation sites - number of pups born alive)/number of implantation sites] x 100

Offspring viability indices:

- Sex ratio at birth = no. of males alive/no. of females alive
- Viability Index at day 1 = (No. of pups born alive/total no. of pups born) x 100
- Viability Index at day 4 = (No. of pups alive days 4/total no. of pups alive day 1) x 100
- Lactation Index = (No. of pups alive day 21/no. of pups alive on dy 4) x 100

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

slightly reduced in males at mid- and high dose

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

slightly reduced in males at mid- and high dose

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not specified

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: slightly reduced in males at mid- and high dose

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

- General appearance, growth, and food intake of parent rats:
No abnormalities of condition or behaviour were observed in any of the groups during the pre-mating, mating or lactation period. At 0.4 and 1% dose level body weights were relatively low in males, while in females the figures of the various groups were very similar. Food intake tended to be decreased in all test groups, but the differencs with the control group were very small.

- Fertility:
All females of the control group and of the 0.16 and 0.4% dose group casted a litter while in the 1.0% dose group only 1 out of 30 females was not fertile. The mean litter size at birth showed some variation amongst the groups, but there were no indications of an adverse effect of the test substance. The resorption quotient of the various groups did not reveal any embryo-toxic properties of the test substance. None of the other parameters examined was affected by the feeding of the test item.

Dose descriptor:

NOAEL

Effect level:

1 other: % in diet

Sex:

male/female

Basis for effect level:

other: only slightly reduced body weight in males of high dose group observed

Clinical signs:

not specified

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

reduced in high dose group

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

The total number of pups born dead in each group was only small, resulting in a high viability index at day 1. The sex ratio (male/female) of the pups at birth tended to decrease with increasing levels of DTB-glycolester in the diet. Mortality of the offspring during lactation, as expressed by the viability index at day 4 and the lactation index, was low and was not adversely affected by the test substance. The mean pup weight at day 1, 4 and 14 was comparable in the various groups, but at day 21 body weights in the high dose group were lower than in controls.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 other: % in diet

Sex:

male/female

Basis for effect level:

other: reduced body weight in high dose group

Reproductive effects observed:

not specified

Conclusions:

HostanoxO 3 was studied for its reprodcutive toxic/teratogenic properties in a one-generation combined chronic toxicity study in male and female Wistar rats. The NOAEL was considered to be 1% (corresponding to 500 mg/kg bw/d; highest dose tested) in diet. None of the fertility/viability parameters examined was adversely affected by the feeding of the test substance. Slightly reduced body weight was observed in males and pups of the high dose group.

Executive summary:

The feeding of DTB-glycolester at dietary levels of 0, 0.16, 0.4 or 1% (corresponding to 0, 70, 200 and 500 mg/kg bw/d considering a diet conversion factor (ppm to mg/kg bw/d) of 20) to rats resulted in slight growth depression and relatively low food intake in male parent rats of the mid and high dose group. The only change observed in the litters consisted of slight growth retardation of the pups in the top dose group during the final stage of the lactation period. The other data on fertility, lactation performance and survival did not reveal any abnormalities.

From the present reproduction study it is therefore concluded that the feeding of the test substance at dietary levels up to 1.0% failed to induce any deleterious effects, other than slight growth depression in parent males at 0.4 and 1.0% and in pups at 1.0%.

The F1 -generation was used for a subsequent chronic toxicity study (130 weeks duration, please refer to IUCLID Chapter 7.5.1). No visceral malformations could be found at the termination of this chronic study and therefore, no teratogenic effects were recorded. Furthermore, the rate of mortality was not affected, the body weight differences of rats of the dose groups with the control animals were smaller than 10% and there were no outstanding differences in food intake observed during the chronic toxicity study performed in the F1 -generation.

The NOAEL was considered to be 1% (corresponding to 500 mg/kg bw/d; highest dose tested) in diet. None of the fertility/viability parameters examined was adversely affected by the feeding of the test substance. Slightly reduced body weight was observed in males and pups of the high dose group.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-06-05 to 2018-11-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016

Deviations:

yes

Remarks:

Two satellite groups (high dose and control) for cross-foster experiment. Exposure controls (i.e. milk plus plasma) of high dose dams involved in cross-fostering experiment.

Principles of method if other than guideline:

- Principle of test:
Two additional satellite groups for cross-foster experiment consisting of 5 dams each for control and high dose group.
- Short description of test conditions:
After littering (PND 1), litters of high dose treated females are cross- fostered to control dams and vice versa. Milk and blood samples from dams involved in cross-foster experiment were collected at lactation days 11-12 for exposure analysis.
- Parameters analysed / observed:
Test item concentration

GLP compliance:

yes

Limit test:

no

Justification for study design:

Data from previous repeated dose studies indicated a possible treatment-related effect of Hostanox O 3 P on pre- and postnatal development. To investigate if this developmental toxicity is caused by intrauterine exposure of the conceptus during gestation and/or postnatal exposure of the pups via maternal milk, two Satellite groups (Group 5 and 6) were included in the current study. These satellite groups were treated in the same manner as Groups 1-4. Group 5 was administered the high dose, and Group 6 served as control receiving the standard powder rodent diet without test item. After littering (PND 1), litters of high dose Group 5 were cross-fostered with litters of control Group 6, and vice versa. On PND 11 or 12, blood and milk samples were taken from the dams for exposure control.

Specific details on test material used for the study:

Chemical name (IUPAC): [Bis[3,3-bis-[4`-hydroxy-3`-tert-butylphenyl) butanoicacid] glycolester]

CAS No.: 32509-66-3

Batch No.: DEF2109009

Manufactured date: 23.06.2014

Expiry date: 22.06.2020

Purity as per Certificate of Analysis : 97.4 area %

Physical appearance: White powder

Storage conditions: Ambient (+15 to +25ºC)

Species:

rat

Strain:

Wistar

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION

- Mixing appropriate amounts with (Type of food) and storage temperature of food:
The test item was mixed without the use of a vehicle, directly with the required amount of powder feed. Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was used. Diets were stored in glass containers until use. Care was taken to avoid contact of the diet containing test item with the plastic lid of the container. When stored at room temperature (under normal laboratory light conditions), diets containing test item were used within 8 days after preparation. When stored at room temperature protected from light, diets containing test item were used within 3 weeks after preparation. When stored in the freezer (≤ -15°C), diets containing test item were prepared one week in advance. Upon removal from the freezer they were acclimatized at room temperature for a minimum of 30 minutes before use.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: All mated females showed evidence of mating within 6 days, except for one control female for which it took 13 days before mating could be confirmed.
- Proof of pregnancy: vaginal plug / sperm in vaginal smear, referred to as Day 0 post coitum
- After 8 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical analyses of dietary preparations were conducted once during the study to assess accuracy and homogeneity. Dietary analyses confirmed that the test diets were prepared accurately and homogenously over the concentration range used in this study.

Duration of treatment / exposure:

Males were exposed for 29 or 34 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period.
Females were exposed for 58 - 71 days, i.e. 14 days prior to mating (to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy, and at least 21 days after delivery, up to and including the day of scheduled necropsy. Females which failed to deliver were treated for 43 days. The single female that was euthanized in extremis was treated for 34 days.

Frequency of treatment:

Continuously by diet.

Dose / conc.:

3 750 ppm (nominal)

Remarks:

equivalent to 250 mg/kg bw/day

Dose / conc.:

7 500 ppm (nominal)

Remarks:

equivalent to 500 mg/kg bw/day

Dose / conc.:

11 250 ppm (nominal)

Remarks:

equivalent to 750 mg/kg bw/day

No. of animals per sex per dose:

Control group: 10 per sex
Low dose group:10 per sex
Mid dose group: 10 per sex
High dose group: 10 per sex
Control group for cross-fostering: 5 females were mated with males from Group 4 (both high dose groups) that have previously shown evidence of mating.
High dose group for cross-fostering: 5 females were mated with males from Group 1 (both control groups) that have previously shown evidence of mating

Control animals:

yes

yes, plain diet

Details on study design:

Dose levels were selected based on the results from two old reproduction toxicity studies (1976 and 1979) with dietary administration of DTB-glycolester in rats.
- In one study diets containing 0, 0.1, 0.4 or 1.6 % DBT-glycolester were applied, where the highest dose of 1.6 % was equal to 800 mg/kg bw/day.
- In a second study diets containing 0, 0.16, 0.4 or 1.0 % DBT-glycolester were applied, where 1.0 % was equal to 500 mg/kg bw/day.

Positive control:

No

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily, beginning during the first administration onwards up to the day prior to necropsy

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of administration, and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 13 and 21.
A terminal weight was recorded on the day of scheduled necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 13 and 21.

- Relative Food consumption for each animal calculated against the body weight for scheduled intervals.

- Compound intake: Calculated as concentration of test item in diet (ppm) against relative food consumption. (mean over means intake [mg test item/kg body weight])

Oestrous cyclicity (parental animals):

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Daily vaginal lavage was performed for all females beginning 14 days prior to treatment for Groups 1-4 and 19 days prior to treatment for Groups 5 and 6 (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. Note: No vaginal lavage was prepared for female nos. 81-90, inclusive, on Day 13 of the pre-mating period.
On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus. This was done for all females, except for the female that had to be euthanized in extremis

Sperm parameters (parental animals):

Parameters examined in male parental generations:
For the testes of all males of Groups 1 and 4, and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. [testis weight, epididymis weight, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology].

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [yes]
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups]

GROSS EXAMINATION OF DEAD PUPS:
[yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.]

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals following completion of the mating period (after a minimum of 28 days of administration)
- Maternal animals:
All surviving females (which delivered): at PND 21-23.
Females which failed to deliver:
- With evidence of mating: post coitum Day 25-27 (Note: One control female (# 48) was euthanized in extremis on postcoitum Day 18)
- Without evidence of mating: 24 days after the last day of the mating period (one high dose female # 83)

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table A and B were prepared for microscopic examination and weighed, respectively [refer also to attachment "209310 20146107 Attachment C, Amendment 8 to SP, Tissue collection and Preservation"] .

Postmortem examinations (offspring):

SACRIFICE
- Pups, younger than 7 days were euthanized by decapitation.
All remaining pups (PND 7-23), except for the two pups per litter selected for blood collection were euthanized by an intraperitoneal injection of sodium pentobarbital (Euthasol® 20%).
The pups selected for blood collection on PND 21-23 were anesthetized using isoflurane followed by exsanguination.

- Unscheduled Deaths - F1-Generation:
Pups that died or were euthanized before scheduled termination were examined externally and sexed (both externally and internally).
Pups found dead during the weekend were fixed in identified containers containing 70% ethanol as they were not necropsied on the same day.
The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

- Scheduled Euthanasia - F1-Generation:
On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation. From two surplus pups per litter, blood was collected, if possible.
All remaining pups were euthanized on PND 21-23. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development. The head (incl. right eye) of pup no. 4 in litter 67 was collected and fixed in 10% buffered formalin due to an external abnormality (missing left eye).
In addition, blood was collected from two pups per litter, and the thyroid from two pups per litter (one male and one female pup) was preserved in 10% buffered formalin. The pups selected for (complete) blood sampling were the same pups as selected for thyroid preservation.


GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGTHS
Thyroids from two pups per litter (one male and one female) was preserved in 10% buffered formalin and prepared for microscopic examination.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons were made for dams without cross-fostered litters:
Group 2 vs. Groups 1, 6 (controls)
Group 3 vs. Groups 1, 6 (controls)
Groups 4, 5 vs. Groups 1, 6 (controls)
The following pairwise comparisons were made for dams with cross-fostered litters:
Groups 4, 5 vs. Groups 1, 6 (controls)

- Parametric:
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).

- Non-Parametric:
Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).

- Incidence:
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Reproductive indices:

For each group, the following calculations were performed.
Group mean values of precoital time and duration of gestation were calculated from individual values of F0-females, the remaining group values were calculated from the total number in each group.

Mating index (%): Number of females mated
--------------------------------------- x 100
Number of females paired

Precoital time: Number of days between initiation of cohabitation and confirmation of mating

Fertility index (%): Number of pregnant females
------------------------------------------ x 100
Number of females mated

Gestation index (%): Number of females with living pups on Day 1
----------------------------------------------------------------- x 100
Number of pregnant females

Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Post-implantation survival index (%): Total number of offspring born
--------------------------------------------------------------x 100
Total number of uterine implantation sites

Offspring viability indices:

Live birth index (%): Number of live offspring on Day 1 after littering
------------------------------------------------------------------- x 100
Total number of offspring born

Percentage live males at First Litter Check (%): Number of live male pups at First Litter Check
-------------------------------------------------------------------- x 100
Number of live pups at First Litter Check

Percentage live females at First Litter Check (%): Number of live female pups at First Litter Check
--------------------------------------------------------------------- x 100
Number of live pups at First Litter Check

Viability index (%): Number of live offspring on Day 4 before culling
--------------------------------------------------------------------- x 100
Number live offspring on Day 1 after littering

Lactation index (%): Number of live offspring on Day 21 after littering
--------------------------------------------------------------------- x 100
Number live offspring on Day 4 (after culling)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related clinical signs were noted in males and females up to 11250 ppm during daily detailed clinical observations.
Incidental findings that were noted in females only included piloerection, alopecia and scabbing. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Dermal irritation (if dermal study):

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

- One female of the 7500 ppm group (no. 65) died at blood sampling before scheduled necropsy on lactation Day 23. At necropsy, dark-red discolouration of the glandular mucosa of the stomach was noted (microscopic correlate: slight congestion). This incidental death was regarded to be related to the blood sampling procedure under anesthesia and was considered unrelated to the treatment with the test item.
- One control female (no. 48) was sacrificed in extremis on Day 18 post-coitum. This animal was noted with scabs on her tail from the second week of treatment onwards. After 34 days, severity of scabbing was increased from slight to moderate and the tail was swollen. During a veterinary inspection performed on the same morning, a wound was observed which showed signs of inflammation (reported as focal erythema at moderate degree) and necrosis. As a relatively large area of the tail was affected (approximately 25% from the tail tip), the wound was considered to be painful and it was decided to euthanize this female for humane reasons. This mortality of a control female was not related treatment with the test item.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- A trend towards lower body weight gain was noted in males at 11250 ppm during the entire observation period. Changes in males were relatively slight, reaching statistical significance after three weeks of treatment only. At the end of the observation period (Day 29), mean body weight for males of the 11250 ppm group was slightly lower (4%) than for the concurrent control group.

- Body weights and body weight gain of treated females remained in the same range as controls during pre-mating and post-coitum. During lactation, body weights were significantly decreased in 11250 ppm females on PND 13 and 21 with values 12% and 11% lower compared to controls. Body weight gain was significantly decreased (7% lower compared to controls) in 11250 ppm females on PND 13 and 21. A similar trend towards decreased body weight and body weight gain was observed for females of the 7500 ppm group (mid dose) during lactation, although not statistically significant. At the end of the lactation period (Day 21), mean body weight for females at 7500 ppm was slightly lower (5%) than for the concurrent control group.

- No treatment-related effects on body weights and body weight gain were noted in males at 3750 and 7500 ppm, and in females at 3750 ppm.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Note: When compared to the pre-mating period, a generally higher food intake (absolute and relative to body weight) was measured in females during the phases of gestation and lactation, with values for the control group within normal range. The observed higher food intake is a normal physiological process. It reflects the increased nutritional need of the dams during the periods of gestation and lactation. Furthermore, from the third week of lactation onwards also their pups start to consume solid feed for themselves.

In females, lower absolute and relative food consumption was observed in all treated groups during post-coitum and lactation (not always statistically significant) with changes compared to the concurrent control group most pronounced in the high dose group. During the last week of the lactation period (Days 13- 21), mean relative food consumption for females at 11250 and 7500 ppm was 40% and 18%, respectively, lower than for the concurrent control group, while recovery to normal values was seen at the lowest dose level.

The slightly lower absolute and relative food consumption observed for males and females at 11250 ppm in the first week of treatment (Week 1 of pre-mating) was considered not a sign of toxicity, but to reflect reduced palatability. Changes compared to the concurrent control group were relatively slight (reaching no statistical significance) and recovery towards normal values was seen in the second week of mating.

All animals were treated with fixed test item concentrations in diet. Mean of means of test item intake over study period (pre-mating, post-coitum) were determined 271, 513 and 809 mg test item/kg body weight for males at dose levels of 3750, 7500, 11250 ppm. Mean of means of test item intake over study period (pre-mating, post-coitum, lactation) were determined 501, 1013 and 1320 mg test item/kg body weight for females at dose levels of 3750, 7500, 11250 ppm.

Food efficiency:

not specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum levels of total T4 in P0-males were not affected by treatment. All values remained within the range of available historical control data.
Note: Assessment of T4 in P0-females and TSH in F0-animals of both sexes was considered not relevant because no treatment-related changes in T4 were noted in P0-males, and no treatment-related changes in thyroid weight and no adverse effects on thyroid histopathology were observed in P0-males and P0-females.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were present in females in the organs which were evaluated because of gross findings. (Note: In this type of repro-screening study only tissues from animals with gross findings are preserved and examined.)
Adrenal glands:
- Hemorrhage/congestion of the zona fasciculata at minimal to marked degree was recorded in adrenal glands of six females at 11250 ppm. This alteration was predominantly present at the inner part of the zona fasciculata.
Thymus:
- Lymphoid depletion was recorded for the females with macroscopic reduced size and was seen at marked degree in the two females at 11250 ppm and at slight degree in one female at 7500 ppm

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Description (incidence and severity):

To investigate if the developmental toxicity observed in the older reproduction study is caused by intrauterine exposure of the conceptus during gestation and/or post-natal exposure of the pups via maternal milk, two Satellite groups (group 5 and 6, consisting of 5 dams each) were included in this study for cross-fostering. The rats were fed diets containing 0, 3750, 7500 and 11250 ppm active ingredient. The additional satellite groups used for cross-fostering were treated in the same manner as Groups 1 to 4 with diets of 0 and 11250 ppm. Group 5 was administered the high dose, and Group 6 served as control receiving the standard powder rodent diet without test item. After littering (PND 1), litters of high dose Group 5 were cross-fostered with litters of control Group 6, and vice versa. On PND 11 or 12, blood and milk samples were taken from the dams used for cross-fostering for exposure control analysis.

- Parameters analysed:
Test item concentration Plasma: ranging from 1650 to 5660 ng/mL
Test item concentration Milk: ranging from 1280 to 5990 ng/mL

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were not considered to have been affected by treatment.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

- Precoital time was considered to be unaffected by treatment. All mated females showed evidence of mating within 6 days, except for one control female for which it took 13 days before mating could be confirmed.

- Number of implantation sites was not considered to be affected by treatment.

- Mating index was not considered to be affected by treatment. The mating indices were 100% for the control, 3750 and 7500 groups each, and 93% for the 11250 ppm group. One high dose female (no. 83) was not mated. This can be seen occasionally in this type of study. As only a single female was affected, group mean value for mating index remained within the available control range, and in the absence of any effect on all other reproductive parameters investigated in this study, this finding was considered to be unrelated to treatment.

- Fertility index was not considered to be affected by treatment. All mated females were pregnant. The fertility indices were 80, 70, 80 and 93% for the control, 3750, 7500 and 11250 ppm groups, respectively. The fertility index of 70% in the 3750 ppm group was below the available historical control range. However, in the absence of a dose-related trend no toxicological relevance was attributed to this finding.
The number of non-pregnant females versus mated females was 3/15, 3/10, 2/10 and 1/14 in the control, 3750, 7500 and 11250 ppm groups, respectively. Since these cases of non-pregnancy showed no dose-related incidence across the dose groups, and given the absence of any reproductive/developmental toxicity, this was not considered to be related to treatment.

Body weight:

- A trend towards lower body weight gain was noted in males at 11250 ppm during the entire observation period. Changes in males were relatively slight, reaching statistical significance after three weeks of treatment only. At the end of the observation period (Day 29), mean body weight for males of the 11250 ppm group was slightly lower (4%) than for the concurrent control group.

- Body weights and body weight gain of treated females remained in the same range as controls during pre-mating and post-coitum. During lactation, body weights were significantly decreased in 11250 ppm females on PND 13 and 21 with values 12% and 11% lower compared to controls. Body weight gain was significantly decreased (7% lower compared to controls) in 11250 ppm females on PND 13 and 21. A similar trend towards decreased body weight and body weight gain was observed for females of the 7500 ppm group (mid dose) during lactation, although not statistically significant. At the end of the lactation period (Day 21), mean body weight for females at 7500 ppm was slightly lower (5%) than for the concurrent control group.

- No treatment-related effects on body weights and body weight gain were noted in males at 3750 and 7500 ppm, and in females at 3750 ppm.

Food consaumption and test item intake:

Note: When compared to the pre-mating period, a generally higher food intake (absolute and relative to body weight) was measured in females during the phases of gestation and lactation, with values for the control group within normal range. The observed higher food intake is a normal physiological process. It reflects the increased nutritional need of the dams during the periods of gestation and lactation. Furthermore, from the third week of lactation onwards also their pups start to consume solid feed for themselves.

In females, lower absolute and relative food consumption was observed in all treated groups during post-coitum and lactation (not always statistically significant) with changes compared to the concurrent control group most pronounced in the high dose group. During the last week of the lactation period (Days 13- 21), mean relative food consumption for females at 11250 and 7500 ppm was 40% and 18%, respectively, lower than for the concurrent control group, while recovery to normal values was seen at the lowest dose level.

The slightly lower absolute and relative food consumption observed for males and females at 11250 ppm in the first week of treatment (Week 1 of pre-mating) was considered not a sign of toxicity, but to reflect reduced palatability. Changes compared to the concurrent control group were relatively slight (reaching no statistical significance) and recovery towards normal values was seen in the second week of mating.

All animals were treated with fixed test item concentrations in diet. Mean of means of test item intake over study period (pre-mating, post-coitum) were determined 271, 513 and 809 mg test item/kg body weight for males at dose levels of 3750, 7500, 11250 ppm. Mean of means of test item intake over study period (pre-mating, post-coitum, lactation) were determined 501, 1013 and 1320 mg test item/kg body weight for females at dose levels of 3750, 7500, 11250 ppm.


Developmental data:

Note: Due to the five extra females for cross-fostering in the control and high dose (11250 ppm) group, the number of females with offspring available for evaluation was 12, 7, 8 and 13 in the control, 3750, 7500 and 11250 ppm groups, respectively. Where relevant, it was distinguished between data obtained from litters not used for cross-fostering and litters used for cross-fostering.

- Gestation Index and Duration:
Gestation index and duration of gestation were not considered to be affected by treatment. In all groups all pregnant females had live offspring (i.e. gestation index: 100%).

- Parturition/Maternal Care:
No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

- Post implantation Survival Index:
The total number of offspring born compared to the total number of uterine implantations was not considered to be affected by treatment. Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 96, 91, 91 and 95% for the control, 3750, 7500 and 11250 ppm groups, respectively.

For the following females, the number of pups was slightly higher than the number of implantations: control female nos. 43, 45 and 47, and high dose female no. 76. This phenomenon is observed from time to time and is caused by normal resorption of these areas during the 21-23 days of lactation. No toxicological relevance was attached to this finding in the current study.

- Litter Size:
Litter size was not considered affected by treatment.
When excluding litters used for cross-fostering, live litter sizes were 11.6, 10.6, 10.3 and 10.3 living fetuses/litter for the control, 3750, 7500 and 11250 ppm groups, respectively.
Litters used for cross-fostering had a live litter size of 10.0 and 11.6 in the control and 11250 ppm group, respectively (after cross-fostering).

- Live Birth Index:
The number of live offspring on Day 1 after littering compared to the total number of offspring born was not considered to be affected by treatment. Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was considered not to be affected by treatment. The live birth indices were 100% for the control, 3750 and 7500 groups each, and 99% for the 11250 ppm group.
One pup of the 11250 ppm group (litter 84; not cross-fostered) was found dead at first litter check. No toxicological relevance was attributed to this single dead pup since the mortality incidence remained within the range considered normal for pups of this age.

- Viability Index:
The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was not considered affected by treatment. Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment.
Without cross-fostering (control, 3750, 7500 and 11250 ppm groups):
One pup each in the control and 7500 ppm groups, and two pups in one litter in the 11250 ppm group went missing (presumably cannibalized) between lactation Days 2 and 4. In the same high dose litter (no. 84) there was also the only one dead pup at first litter check in this study (see previous section); the remaining two pups survived until scheduled necropsy on lactation Day 23. Occasionally, a litter in poor condition is seen in rats of this age and strain. As all remaining litters in the high dose group were completely normal, no toxicological relevance was attached to this finding. Viability indices were 99, 100, 99 and 98% for the control, 3750, 7500 and 11250 ppm groups, respectively. All these values remained well within the normal rage of biological variation.
With cross-fostering (control and 11250 ppm groups):
No cross-fostered pups were found dead/missing. Viability index was 100% for both the control and 11250 ppm group.

-Lactation Index:
The number of live offspring on Day 21 after littering compared to the number of live offspring on Day 4 (after culling) was not considered to be affected by treatment.
Without cross-fostering (control, 3750, 7500 and 11250 ppm groups):
One pup in the 3750 ppm was found dead on lactation Day 16, resulting in a lactation index of 98% for this low dose group compared to 100% for all remaining groups. In the absence of a dose-related trend no toxicological relevance was attributed to this finding.
After cross-fostering (control and 11250 ppm groups):
The lactation index was 100% for both the control and 11250 ppm group.

Key result

Dose descriptor:

NOAEL

Effect level:

7 500 ppm (nominal)

Based on:

test mat. (total fraction)

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

11 250 ppm

System:

immune system

Organ:

adrenal glands

thymus

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

not specified

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment.
One pup of the 7500 ppm group (pup 4/litter 67) was observed with one eye missing from lactation Day 21 onwards. This isolated finding at the mid dose level was considered unrelated to treatment with the test item.
The nature and incidence of other clinical signs (blue spots and scabs on different parts of the body, missing tail apex, lean appearance and/or less milk) remained within the range considered normal for pups of this age, and were, therefore, not considered to be toxicologically relevant.

Dermal irritation (if dermal study):

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

- Post implantation Survival Index:
The total number of offspring born compared to the total number of uterine implantations was not considered to be affected by treatment. Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 96, 91, 91 and 95% for the control, 3750, 7500 and 11250 ppm groups, respectively.

For the following females, the number of pups was slightly higher than the number of implantations: control female nos. 43, 45 and 47, and high dose female no. 76. This phenomenon is observed from time to time and is caused by normal resorption of these areas during the 21-23 days of lactation. No toxicological relevance was attached to this finding in the current study.

- Litter Size:
Litter size was not considered affected by treatment.
When excluding litters used for cross-fostering, live litter sizes were 11.6, 10.6, 10.3 and 10.3 living fetuses/litter for the control, 3750, 7500 and 11250 ppm groups, respectively.
Litters used for cross-fostering had a live litter size of 10.0 and 11.6 in the control and 11250 ppm group, respectively (after cross-fostering).

- Live Birth Index:
The number of live offspring on Day 1 after littering compared to the total number of offspring born was not considered to be affected by treatment. Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was considered not to be affected by treatment. The live birth indices were 100% for the control, 3750 and 7500 groups each, 99% for the 11250 ppm group.
One pup of the 11250 ppm group (litter 84; not cross-fostered) was found dead at first litter check. No toxicological relevance was attributed to this single dead pup since the mortality incidence remained within the range considered normal for pups of this age.

- Viability Index:
The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was not considered affected by treatment. Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment.
Without cross-fostering (control, 3750, 7500 and 11250 ppm groups):
One pup each in the control and 7500 ppm groups, and two pups in one litter in the 11250 ppm group went missing (presumably cannibalized) between lactation Days 2 and 4. In the same high dose litter (no. 84) there was also the only one dead pup at first litter check in this study (see previous section); the remaining two pups survived until scheduled necropsy on lactation Day 23. Occasionally, a litter in poor condition is seen in rats of this age and strain. As all remaining litters in the high dose group were completely normal, no toxicological relevance was attached to this finding. Viability indices were 99, 100, 99 and 98% for the control, 3750, 7500 and 11250 ppm groups, respectively. All these values remained well within the normal rage of biological variation.
With cross-fostering (control and 11250 ppm groups):
No cross-fostered pups were found dead/missing. Viability index was 100% for both the control and 11250 ppm group.

-Lactation Index:
The number of live offspring on Day 21 after littering compared to the number of live offspring on Day 4 (after culling) was not considered to be affected by treatment.
Without cross-fostering (control, 3750, 7500 and 11250 ppm groups):
One pup in the 3750 ppm was found dead on lactation Day 16, resulting in a lactation index of 98% for this low dose group compared to 100% for all remaining groups. In the absence of a dose-related trend no toxicological relevance was attributed to this finding.
After cross-fostering (control and 11250 ppm groups):
The lactation index was 100% for both the control and 11250 ppm group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Without cross-fostering (control, 3750, 7500 and 11250 ppm groups):
At birth, mean pup body weight for male pups, female pups and both sexes combined was similar between the control and all treated groups. However, a trend towards lower body weights was noted in male and female pups of the 11250 ppm and 7500 ppm groups from PND 4 and PND 13, respectively, onwards, reaching statistical significance on PND 13 and PND 21 for pups at 11250 ppm (relative difference to concurrent control group: -19% and -34% for sexes combined) and PND 21 for pups at 7500 ppm (relative difference to concurrent control group: -16 % for sexes combined). At the end of observation period (PND 21), mean body weights of pups in both the 7500 and 11250 ppm group (males:41.2 and 33.7 gram, respectively; females 41.4 and 32.4 gram, respectively) were below the available historical range.

With cross-fostering (control and 11250 ppm groups):
At birth, mean pup body weight for male pups, female pups and both sexes combined was similar between the control and 11250 ppm groups. However, from lactation Day 4 onwards, a trend towards lower body weights was noted in male and female pups from control females, cross-fostered to 11250 ppm females, reaching statistical significance from PND 7 onwards. On PND 13 and PND 21, mean body weights for male and female pups (males: 22.9 and 27.1 gram, respectively; females: 22.3 and 27.2 gram, respectively) were clearly below the available historical range. At the end of the observation period (PND 21), mean body weight for sexes combined was 45% lower compared to the mean body weight of pups from 11250 ppm females, cross-fostered to control females. It should be noted that mean body weight of latter cross-fostered pups (from the 11250 ppm group females to control females not treated), was similar to that of non-cross-fostered control pups or slightly higher throughout the observation period.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum T4 levels in male and female pups at PND 21-23 were judged to be unaffected by treatment.
After cross-fostering to the 11250 ppm group, both pups in litter 75 (male: 6.90 µg/dL; female: 6.16 µg/dL), one male pup (litter 76: 6.33 µg/dL) and one female pup (litter 71: 7.19 µg/dL) had a relatively high value for total T4. As no comparable trend was observed for the pup of the opposite sex in the same litter (except for litter 75) or any of the pups in the high dose group without cross-fostering, the incidentally higher individual values were considered not to be toxicologically relevant.

Note: Assessment of T4 for PND 4 pups and TSH for PND 21-23 pups was considered not relevant because no treatment-related changes in T4 were noted in pups at PND 21-23.

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

Anogenital distance:
Anogenital distance (absolute and normalized for cube root of body weight) in male and female pups was not considered to be affected by treatment. All values remained well within the available control range.
Areola/Nipple Retention:
Treatment up to 11250 ppm had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment.
The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore not considered to be related to treatment.

Histopathological findings:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

- Litter Size:
Litter size was not considered affected by treatment.
When excluding litters used for cross-fostering, live litter sizes were 11.6, 10.6, 10.3 and 10.3 living fetuses/litter for the control, 3750, 7500 and 11250 ppm groups, respectively.
Litters used for cross-fostering had a live litter size of 10.0 and 11.6 in the control and 11250 ppm group, respectively (after cross-fostering).

- Live Birth Index:
The number of live offspring on Day 1 after littering compared to the total number of offspring born was not considered to be affected by treatment. Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was considered not to be affected by treatment. The live birth indices were 100% for the control, 3750 and 7500 groups each, and 99% for the 11250 ppm group.
One pup of the 11250 ppm group (litter 84; not cross-fostered) was found dead at first litter check. No toxicological relevance was attributed to this single dead pup since the mortality incidence remained within the range considered normal for pups of this age.

- Viability Index:
The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was not considered affected by treatment. Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment.
- Without cross-fostering (control, 3750, 7500 and 11250 ppm groups):
One pup each in the control and 7500 ppm groups, and two pups in one litter in the 11250 ppm group went missing (presumably cannibalized) between lactation Days 2 and 4. In the same high dose litter (no. 84) there was also the only one dead pup at first litter check in this study (see previous section); the remaining two pups survived until scheduled necropsy on lactation Day 23. Occasionally, a litter in poor condition is seen in rats of this age and strain. As all remaining litters in the high dose group were completely normal, no toxicological relevance was attached to this finding. Viability indices were 99, 100, 99 and 98% for the control, 3750, 7500 and 11250 ppm groups, respectively. All these values remained well within the normal rage of biological variation.
- With cross-fostering (control and 11250 ppm groups):
No cross-fostered pups were found dead/missing. Viability index was 100% for both the control and 11250 ppm group.

- Lactation Index:
The number of live offspring on Day 21 after littering compared to the number of live offspring on Day 4 (after culling) was not considered to be affected by treatment.
- Lactation Index without cross-fostering (control, 3750, 7500 and 11250 ppm groups):
One pup in the 3750 ppm was found dead on lactation Day 16, resulting in a lactation index of 98% for this low dose group compared to 100% for all remaining groups. In the absence of a dose-related trend no toxicological relevance was attributed to this finding.
- Lactation Index after cross-fostering (control and 11250 ppm groups):
The lactation index was 100% for both the control and 11250 ppm group.

- Body weight without cross-fostering (control, 3750, 7500 and 11250 ppm groups):
At birth, mean pup body weight for male pups, female pups and both sexes combined was similar between the control and all treated groups. However, a trend towards lower body weights was noted in male and female pups of the 11250 ppm and 7500 ppm groups from PND 4 and PND 13, respectively, onwards, reaching statistical significance on PND 13 and PND 21 for pups at 11250 ppm (relative difference to concurrent control group: -19% and -34% for sexes combined) and PND 21 for pups at 7500 ppm (relative difference to concurrent control group: -16 % for sexes combined). At the end of observation period (PND 21), mean body weights of pups in both the 7500 and 11250 ppm group (males:41.2 and 33.7 gram, respectively; females 41.4 and 32.4 gram, respectively) were below the available historical range***.

- Body weight with cross-fostering (control and 11250 ppm groups):
At birth, mean pup body weight for male pups, female pups and both sexes combined was similar between the control and 11250 ppm groups. However, from lactation Day 4 onwards, a trend towards lower body weights was noted in male and female pups from control females, cross-fostered to 11250 ppm females, reaching statistical significance from PND 7 onwards. On PND 13 and PND 21, mean body weights for male and female pups (males: 22.9 and 27.1 gram, respectively; females: 22.3 and 27.2 gram, respectively) were clearly below the available historical range***. At the end of the observation period (PND 21), mean body weight for sexes combined was 45% lower compared to the mean body weight of pups from 11250 ppm females, cross-fostered to control females. It should be noted that mean body weight of latter cross-fostered pups (from the 11250 ppm group females to control females not treated), was similar to that of non-cross-fostered control pups or slightly higher throughout the observation period.

***[Historical control data for body weights of pups, Wistar Han rats (period 2017 - 2018):
Pup weight Male pups on PND 13 (gram) mean = 31, P5 - P95 = 27.0 – 34.7 (n=258)
Pup weight Male pups on PND 21 (gram) mean = 51, P5 - P95 = 43.0 – 59.0 (n=350)
Pup weight Female pups on PND 13 (gram) mean = 30, P5 - P95 = 26.7 – 34.4 (n=245)
Pup weight Female pups on PND 21 (gram) mean = 50, P5 - P95 = 43.0 – 58.0 (n=320)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

3 750 ppm (nominal)

Based on:

test mat. (total fraction)

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Reproductive effects observed:

no

Conclusions:

Parental data obtained form an OECD 421 feeding study revealed signs of toxicity at 11250 ppm and 7500 ppm in P0 generation. These consisted of reduced body weight/body weight gain (both sexes), reduced food consumption (females only), and gross findings and microscopic alterations recorded in the adrenal glands (hemorrhage/congestion diffuse cystic degeneration of zona fasciculata, enlargement and/or discoloration) and thymus (lymphoid depletion, reuced size) in females. Significant reduced body weight gain was observed in dams of the high dose group during the course of lactation period at PND 13 and 21. No treatment-related changes in any of the remaining parameters investigated in this study (i.e. viability, clinical appearance, T4 thyroid hormone levels, and organ weights) were noted.
No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, pre-coital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs). Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was not affected. Viability of offspring was not affected by treatment with test substance.

The number of live offspring on Day 21 after littering compared to the number of live offspring on Day 4 (after culling) was not considered to be affected by treatment.

Reduced body weight gain of male and female pups was observed at 7500 and 11250 ppm reaching statistical significance on PND 13 and PND 21 for pups at 11250 ppm. Birth weight of all pups at all dose levels were in the same range as the concurrent control group, indicating that developmental toxicity observed in this study was caused post-natal. This is confirmed by the finding that pups from control females (i.e. females that had not been administered test item), cross-fostered on PND 1 to 11250 ppm females showed a comparable effect of body weight gain during lactation, while pups from 11250 ppm females, cross-fostered on PND 1 to control females had normal body weights/body weight gain. Thus, it is assumed, that pups cross-fostered to high dose females were exposed to the test item by lactational transfer in breast milk. This was confirmed by the results of bioanalysis in plasma and milk samples derived from the dams used for cross-fostering. Based on this observation, it is assumed that effects on reduced body weight gain seen in pups from dams treated with 11250 ppm are caused by lactational transfer of test item in breast milk and not intra-uterine.

No treatment-related changes were noted in any of the remaining parameters for F1-generation investigated in this study (i.e. viability, clinical appearance, sex-ratio, anogenital distance, areola/nipple retention, T4 thyroid hormone levels). No macroscopic findings were noted among pups, that were considered to be related to treatment.

Executive summary:

To investigate if the developmental toxicity observed in the older reproduction study is caused by intrauterine exposure of the conceptus during gestation and/or post-natal exposure of the pups via maternal milk, two Satellite groups (group 5 and 6, consisting of 5 dams each) were included in an OECD 421 guideline study. The rats were fed diets containing 0, 3750, 7500 and 11250 ppm active ingredient.The additional satellite groups used for cross-fostering were treated in the same manner as Groups 1 to 4 with diets of 0 and11250 ppm. Group 5 was administered the high dose, and Group 6 served as control receiving the standard powder rodent diet without test item. After littering (PND 1), litters of high dose Group 5 were cross-fostered with litters of control Group 6, andvice versa. On PND 11 or 12, blood and milk samples were taken from the dams for exposure control.

Signs of toxicity were noted at 11250 ppm and 7500 ppm in P0 generation. These consisted of reduced body weight/body weight gain (both sexes), reduced food consumption (females only), and gross findings and microscopic alterationsrecorded in the adrenal glands (up to marked degree of hemorrhage/congestion and diffuse cystic degeneration of the zona fasciculata, correlating to enlargement and/or discoloration) and thymus (lymphoid depletion at marked degree, correlating to reduced size) in females.

In lactating females at 11250 ppm, reduced body weight gain (7% lower compared to controls) was noted on post-natal day (PND) 13 and 21 resulting in body weights that were respectively 12% and 11% lower compared to controls in the same period. A similar trend towards decreased body weight and body weight gain was observed for lactating females of the 7500 ppm group (mid dose), although not statistically significant.

Lower absolute and relative food consumption was observed in all treated groups during post-coitum and lactation (not always statistically significant) with changes compared to the concurrent control group most pronounced in the high dose group. During the last week of the lactation period (PND 13- 21), mean relative food consumption for females at 11250 ppm and 7500 ppm was 40% and 18%, respectively, lower than for the concurrent control group, while recovery to normal values was seen at the lowest dose level of 3750 ppm.

Test item intake (mean of means) over study period (pre-mating, post-coitum) were determined for males to be 271, 513 and 809 mg test item/kg body weight at dose levels of 3750, 7500, 11250 ppm. For females mean of means of test item intake over study period (pre-mating, post-coitum, lactation) were determined to be 501, 1013 and 1320 mg test item/kg body weight at dose levels of 3750, 7500, 11250 ppm. Bioanalysis for exposure control in plasma during lactation (days 11 or 12) in all high dose females used for cross-fostering revealed that these females were exposed to the test substance in considerable high doses (ranging from 1650 to 5660 ng/mL). Comparable concentrations were also measured in milk of these dams (ranging from 1280 to 5990 ng/mL).

No treatment-related changes were noted in any of the remaining parameters investigated in this study (i.e. viability, clinical appearance, T4 thyroid hormone levels, and organ weights).

No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, pre-coital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

The total number of offspring born compared to the total number of uterine implantations was not considered to be affected by treatment. Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 96, 91, 91 and 95% for the control, 3750, 7500 and 11250 ppm groups, respectively.

Birth weight of all pups at all dose levels were in the same range as the concurrent control group.

Viability of offspring was not affected by treatment with test substance. Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was 99, 100, 99 and 98% for the control, 3750, 7500 and 11250 ppm groups, respectively. For cross-fostered pups the Day 4 viability index was 100% for both the control and 11250 ppm group.

The number of live offspring on Day 21 after littering compared to the number of live offspring on Day 4 (after culling) was not considered to be affected by treatment. One pup in the 3750 ppm was found dead on lactation Day 16, resulting in a lactation index of 98% for this low dose group compared to 100% for all remaining groups. The lactation index for pups after cross-fostering was 100% for both the control and 11250 ppm group. 

Reduced body weight gain of male and female pups was observed at 7500 and 11250 ppm. A trend towards lower body weight gains was noted in male and female pups of the 11250 ppm and 7500 ppm groups from respectively PND 4 and PND 13 onwards, reaching statistical significance on PND 13 and PND 21 for pups at 11250 ppm group (relative difference to concurrent control group: -19% and -34% for sexes combined) and PND 21 for pups at 7500 ppm group (relative difference to concurrent control group: -16 % for sexes combined). 

Birth weight of all pups at all dose levels were in the same range as the concurrent control group, indicating that developmental toxicity observed in this study was caused post-natal. This is confirmed by the finding that pups from control females (i.e. females that had not been administered test item), cross-fostered on PND 1 to 11250 ppm females showed a comparable effect of body weight gain during lactation, while pups from 11250 ppm females, cross-fostered on PND 1 to control females had normal body weights/body weight gain. Thus, it is assumed, that pups cross-fostered to high dose females were exposed to the test item by lactational transfer in breast milk as was confirmed by the results of bioanalysis in plasma and milk samples derived from the dams. Based on this observation, it is assumed that effects on body weight gain seen in pups from dams treated with 11250 ppm are caused by lactational transfer of test item in breast milk.

No treatment-related changes were noted in any of the remaining parameters for F1-generation investigated in this study (i.e. viability, clinical appearance, sex-ratio, anogenital distance, areola/nipple retention, T4 thyroid hormone levels). No macroscopic findings were noted among pups, that were considered to be related to treatment.

The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age and were therefore not considered to be related to treatment.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Well performed and reported non-guideline study with scientific sound design and sufficient reporting (key study). Reporting of supporting study not sufficient. OCED 421 study is a guideline conform study.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Quality of whole database:

In accordance with column 2 of REACH Annexes VIII and IX, the repeated dose toxicity study, as required in section 8.6.1 of Annex VIII and in section 8.6.2 of Annex IX, does not need to use the inhalation route because exposure of human via inhalation, especially in a higher extent than via oral application as performed in the animal studies, is considered unlikely taking into account the vapour pressure/physical form of the substance, and intended use pattern (embedded in polymeric matrices).

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Quality of whole database:

In accordance with column 2 of REACH Annexes VIII and IX, the repeated dose toxicity study, as required in section 8.6.1 of Annex VIII and in section 8.6.2 of Annex IX, does not need to use the dermal route because - no primary systemic effects or other evidence of absorption were observed in skin and eye irritation studies in rabbits as well as in the local lymph node assay in mice - Due to the combination of its molecular weight (795 g/mol) and the extent of the molecular structure it is unlikely that higher amounts than tested in a repeated oral toxicity study will be systemically available via the intact skin barrier.

Additional information

An OECD 421 guideline conform study is available where rats were fed diets containing 0, 3750, 7500 and 11250 ppm active ingredient (corresponding to nominal dose levels of 0, 250, 500 and 750 mg/kg bw/d, respectively). Signs of toxicity were noted at 11250 ppm and 7500 ppm in P0 generation. These consisted of reduced body weight/body weight gain (both sexes), reduced food consumption (females only), and gross findings and microscopic alterations recorded in the adrenal glands (hemorrhage/congestion and diffuse cystic degeneration of the zona fasciculata, enlargement and/or discoloration) and thymus (lymphoid depletion, reduced size) in females.

No treatment-related changes were noted in any of the remaining parameters investigated in this study (i.e. viability, clinical appearance, T4 thyroid hormone levels, and organ weights)

No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, pre-coital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

The total number of offspring born compared to the total number of uterine implantations was not considered to be affected by treatment. Birth weight of all pups of all dose levels were in the same range as the concurrent control group. A trend towards lower body weight gains was noted in male and female pups of the 11250 ppm and 7500 ppm groups from respectively PND 4 and PND 13 onwards, reaching statistical significance on PND 13 and PND 21 for pups at 11250 ppm group and on PND 21 for pups at 7500 ppm group.

No treatment-related changes were noted in any of the remaining parameters for F1-generation investigated in this study (i.e. viability, clinical appearance, sex-ratio, anogenital distance, areola/nipple retention, T4 thyroid hormone levels). No macroscopic findings were noted among pups, that were considered to be related to treatment.

Effects on developmental toxicity

Description of key information

A developmental toxicity study according to OECD 414 was performed with the test item. Hostanox O 3 P was applied orally (by gavage) to presumed pregnant rats once daily from GD 5 to 19 at the dose levels of 100, 300 and 1000 mg/kg/day for low (G2), mid (G3) and high (G4) dose group rats, respectively. Rats in the vehicle control (G1) group received the vehicle (sunflower oil) alone. Caesarean section was performed for all rats on GD 20. The following changes were observed:

1. Maternal effects:

·        Clinical symptoms:

Reddish vaginal discharge observed in 67% of the high dose animals (1000 mg/kg bw/d)

·          Pathology:

Gross pathological changes of enlarged adrenals at 300 and 1000 mg/kg bw/d and small / not prominent thymus at 1000 mg/kg bw/d.

·        Body weight:

At 1000 mg/kg/day, the mean body weight was significantly lowerat GD 20 (including gravid uterus) with 26% reduction compared to control group.

At 300 mg/kg/day, the maternal body weight gains were significantly lower during entire gestation period GD 0-20 (-17%) as compared to vehicle control group.

·        Food consumption:

25-48% reduced food consumption at 1000 mg/kg bw/d from gestation day 5-8, which is treatment day 1-3, until study termination. The significantly decreased food consumption is considered to be caused by the above described pathological changes of the adrenals and indicates maternal toxicity.

·        Other maternal data:

At 300 and 1000 mg/kg/day, the gravid uterine weights were significantly lower (-22% and -84%, respectively) and there wassignificant increase in late resorptions and post implantation loss. There was significant increase in dams with any resorption and dams with complete resorptions at 300 and 1000 mg/kg/day.

2. Embryotoxic effects:

·        Observed main effect:

Significantly increased number of “late resorptions/post implantation loss” in the mid and high dose group.

These observations indicate a mode of action which is synchronous with the reduced maternal body weight gain and, therefore, can be considered as a secondary effect of the systemic effects.

·        70% less litters at 1000 mg/kg bw/d (secondary effect as discussed above).

·        70% reduced litter size in the high dose group and 15% reduced litter size in the mid dose groupcompared to the control group (secondary effect as discussed above).

·        25% decreased mean fetal body weight at 1000 mg/kg bw/dcompared to the control group (secondary effect as discussed above).

·        No visceral malformationsobserved.

·        No skeletal malformationsobserved.

Based on the reported data the NOAEL (maternal) and NOAEL(developmental) is derived at 100 mg/kg bw/d.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-06-09 to 2017-08-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted on January 22, 2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Chemical name (IUPAC): [Bis[3,3-bis-[4`-hydroxy-3`-tert-butylphenyl) butanoicacid] glycolester]

CAS No.: 32509-66-3

Batch No.: DEF2109009

Manufactured date: 23.06.2014

Expiry date: 22.06.2020

Purity as per Certificate of Analysis : 97.4 area %

Physical appearance: White powder

Storage conditions: Ambient (+15 to +25ºC)

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Vivo Bio Tech Ltd, Sy # 349/A, Pregnapur-502311,Gajwel Mandal, Medak District, Telangana
- Age at study initiation: 13 to 14 weeks
- Weight at study initiation:
Mean body weight (g) Body weight range
G1 : 212.38 ± 13.74 192.21 to 239.45 g
G2 : 212.41 ± 13.80 193.21 to 242.46 g
G3 : 212.42 ± 13.71 193.86 to 239.28 g
G4 : 212.38 ± 14.18 190.26 to 241.21 g

- Housing: standard polysulfone rat cages (size: Length 425 mm x Breadth 266 mm x Height 185 mm) with stainless steel top grill having facilities for pellet food and drinking water in polycarbonate bottle with stainless steel sipper tube
- Diet: Teklad Certified (2014C) Global 14 % Protein Rodent Maintenance Diet - Pellet (Certified) manufactured by Envigo (previously referred to as Harlan Laboratories), P.O.Box 44220, Madison Wi 53744-4220;ad libitum
- Water: yes Deep bore-well water passed through activated charcoal filter and exposed to UV rays in ‘Aquaguard’ on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd., Mumbai 400 001, India; ad libitum
- Acclimation period: 5 days; Females used in this study were nulliparous and non-pregnant.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24 °C,
- Humidity (%): 65 – 68 %
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2016-06-09 To: 2016-07-05 - 2016-07-08 (sacrifice)

Route of administration:

oral: gavage

Vehicle:

other: sunflower oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Test item forms good suspension in sunflower oil as indicated by the study sponsor and the same vehicle was used in the previous studies carried out by study sponsor and hence the same vehicle is selected for the present study.

The dose formulations were prepared at an interval of 3 to 4 days and were used within the established stability period. The prepared dose formulation when not in use were stored in the experimental room.
The homogeneity of the Hostanox O 3 P formulation during treatment/sampling was maintained by constant stirring using a magnetic stirrer.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulations were analyzed for active ingredient concentration (a.i.) and homogeneity in duplicate sets at the initiation of treatment and at termination of treatment period.The prepared formulations were sampled in duplicate sets. One set was used for analysis and another was kept as back up set which was stored in the experimental room. For each set, duplicate samples were drawn from top, middle and bottom layers of each dose formulation. In case of control, duplicate samples from middle layer were drawn. The test item Hostanox O 3 P in the dose formulations was determined using High Performance Liquid Chromatograph (HPLC) with UV Detector. Dose formulation samples were analyzed as per the Analytical method validated under Advinus Study No.G11434. The results of analysis of formulations were within the acceptable limits. The test item was found to be homogeneous in all the dose formulation samples which met the acceptance limits of variation (85 to 115%) from the claimed concentrations and % RSD was less than 10 %.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Proof of pregnancy: If there was presence of sperm in a vaginal smear or if of vaginal plug was observed in the morning, the animal was considered to be mated. This day was considered as Day ‘0’ of gestation.

Duration of treatment / exposure:

gestation day 5 to gestation day 19

Frequency of treatment:

once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

G1, vehicle control group

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

G2, low dose

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

G3, mid dose

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

G4, high dose

No. of animals per sex per dose:

24 pregnant females

Control animals:

yes, concurrent vehicle

Details on study design:

Dose levels were considered to be best choice based on the outcome of available repeated dose studies.
Based on the body weight, day ‘0’ pregnant rats were allotted a serial number in the ascending order. These rats were then assigned to the groups starting from control to treatment groups and then in the reverse order of dose groups to control.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes / No / No data
- Time schedule: Observations for clinical signs were performed twice a day - pre dose and post dose (within 1-2 hours of administration) during treatment days and once on non-treatment days.
- Cage side observations checked in table.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each rat was observed twice daily for morbidity and mortality i.e., once in the morning and once in the afternoon.

BODY WEIGHT: Yes
- Time schedule for examinations: All females included in the study were weighed on gestation days 0, 3, 5, 8, 11, 14, 17 and 20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/rat/day: Yes

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

POST-MORTEM EXAMINATIONS: Yes / No / No data
- Sacrifice on gestation day 20
- Organs examined:
The gravid uterus along with the cervix including ovaries was excised, weighed and immediately examined.

Ovaries and uterine content:

The following maternal data were recorded.
a. Pregnancy status
b. Gravid uterine weight
c. No. of corpora lutea
d. No. of implantation sites
e. No. of early resorptions
f. No. of late resorptions

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:

The data on maternal body weight, body weight change, gravid uterine weight, corrected body weight on gestation day 20 (carcass weight), corrected body weight gain, maternal food consumption, number of corpora lutea, number of implantations, total number of fetuses, male and female fetus number and weight including combined sex was analyzed using ANOVA model, after testing for homogeneity of intra group variance using Levene’s test. When intra group variances were heterogeneous, ANOVA was performed after suitable transformation of data. Dunnett’s pairwise comparison of the treated group means with the control group mean was performed when the group differences were found significant.

Incidences of pre-implantation loss, post implantation loss, number of early and late resorptions were analyzed using Kruskal Wallis test.

Overall percentage of minor external, visceral and skeletal malformations, Sex ratio, number of dams with any resorptions and number of dams with complete resorptions were analyzed using 2 X 2 Contingency Table.

Indices:

MATERNAL DATA:
Maternal Parameters and Formula used

> Mean number of corpora lutea

Total no. of CL
= ----------------------------------------
Total no. of pregnant animals

> Mean number of implantations/group

Total no. of implantations
= ----------------------------------------
Total no. of pregnant animals

> Embryonic resorption index (%)

No. of early resorptions
= --------------------------------- x 100
No. of implantations

> Fetal resorption index (%)

No. of late resorptions
= -------------------------------- x 100
No. of implantations

> Pre-implantation loss per group (%)

No. of CL - No. of implantations
=----------------------------------------------- x 100
No. of CL

> Post-implantation loss per group (%)

No. of (early + late) resorptions
= --------------------------------------------- x 100
Total no. of implantations

> Implantation index (%)

No. of implantations sites
= ------------------------------------- x 100
No. of corpora lutea

> Corrected body weight (Carcass weight)

= Terminal body weight (body weight on GD20) - unopened uterine weight.

> Corrected body weight gain

= Corrected body weight – body weight on GD5

LITTER DATA:
Litter Parameters and Formula used

> Mean litter size per group

Total No. of fetuses
= ---------------------------------
Total No. of pregnant animals

> Percentage of abnormal fetuses

No. of abnormal fetuses
= ---------------------------------- x 100
Total No. of fetuses

> Percentage of live fetuses (Live fetus index)

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

> Percentage of dead fetuses (Dead fetus index)

No. of dead fetuses
= ----------------------------- x 100
Total No. of fetuses

> Sex ratio (F : M)

Number of females
= ---------------------------
Number of males

Historical control data:

The results of the study were discussed taking into consideration the historical data of this lab.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 300 mg/kg/day, there was one incidence of poorly formed faeces containing mostly water in one dam (Rs4625) on GD 20.

At 1000 mg/kg/day, reddish discharge from vagina was observed in most of the rats between GD 13 to 20 which can be correlated to resorptions noted at caesarean section.

Dermal irritation (if dermal study):

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights were unaffected by treatment at 100 and 300 mg/kg bw/day.
At 1000 mg/kg/day, the mean body weight was significantly lower on GD 11 (-5%), 14 (-11%), 17 (-18%) and 20 (-26%) as compared to vehicle control group.

At 100 mg/kg/day, the maternal body weight gains were comparable to vehicle control group.
At 300 mg/kg/day, the maternal body weight gains were significantly lower during GD 14-17 (- 31%), during treatment period GD 5-20 (-22%) and entire gestation period GD 0-20 (-17%) as compared to vehicle control group. The body weight change corrected for gravid uterine weight was lower at 300 mg/kg/day (81%) compared to control.

At 1000 mg/kg/day, there was a significant reduction in maternal body weight gain during GD 5-8 (-66%), GD 8-11 (-71%), GD 11-14 (-157%), GD 14-17 (-87%) and GD 17-20 (-118%). The maternal body weight gains were also significantly lower during treatment period GD 5-20 (-102%) and entire gestation period GD 0-20 (-87%) including the corrected body weight gains (-172%) as compared to vehicle control group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 100 and 300 mg/kg/day, the maternal food consumption was comparable to vehicle control group.

At 1000 mg/kg/day, there was a significant reduction in maternal food consumption during GD 5-8 (-25%), GD 8-11 (-41%), GD 11-14 (-48%), GD 14-17 (-31%) and GD 17-20 (-34%). The maternal food consumption was also significantly lower during treatment period GD 5-20 (-36%) and entire gestation period GD 0-20 (-26%) as compared to vehicle control group.
The significant reduction in food consumption at 1000 mg/kg/day was considered to be treatment-related and indicates maternal toxicity.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

There were no gross pathological findings in control and 100 mg/kg/day dose group.

At 300 mg/kg/day, one dam (Rs4630) showed bilaterally enlarged adrenals.

At 1000 mg/kg/day, there were incidences of thymus small (10/24)/not prominent (9/24) and bilaterally enlarged adrenals (12/24).

The gross pathological changes of enlarged adrenals at 300 and 1000 mg/kg/day and small / not prominent thymus at 1000 mg/kg/day are indicative of maternal stress at these doses.

There were no abnormalities during gross evaluation of placenta.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

At 300 and 1000 mg/kg/day, there were treatment-related significant reduction in maternal body weight gains during the treatment period GD 5-20 (-22% and -102%, respectively) and entire gestation period GD 0-20 (-17% and -87%, respectively). The body weight change corrected for gravid uterine weight was lower at 300 mg/kg/day (81%) and was significantly lower at 1000 mg/kg/day (-172%) compared to controls, indicating maternal toxicity.

The significant reduction in food consumption at 1000 mg/kg/day was considered to be treatment-related and indicates maternal toxicity.

The gross pathological changes of enlarged adrenals at 300 and 1000 mg/kg/day and small / not prominent thymus at 1000 mg/kg/day are indicative of maternal stress at these doses.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

Mean numbers of pre-implantation losses in all treated groups were comparable to the vehicle control group.
There was significant increase in post-implantation loss at 300 and 1000 mg/kg/day.

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

There were two dams out of 24 with total resorptions observed at the 300 mg/kg /day group.
There was significant increase in dams with complete resorptions at 1000 mg/kg/day (17/24).

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

Maternal data parameters comprising of early resorptions in all treated groups were comparable to the vehicle control group.
There was significant increase in late resorptions at 300 and 1000 mg/kg/day.

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Pregnancy duration is not relevant for the study design of the OECD 414 study.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): Pregnancy duration is not relevant for the study design of the OECD 414 study.

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

At 300 and 1000 mg/kg/day, there were treatment-related significant reduction in maternal body weight gains during the treatment period GD 5-20 (-22% and -102%, respectively) and entire gestation period GD 0-20 (-17% and -87%, respectively). The body weight change corrected for gravid uterine weight was lower at 300 mg/kg/day (81%) and was significantly lower at 1000 mg/kg/day (-172%) compared to controls, indicating maternal toxicity.

The significant reduction in food consumption at 1000 mg/kg/day (-28%) compared to controls was considered to be treatment-related and indicates maternal toxicity.

The gross pathological changes of enlarged adrenals at 300 and 1000 mg/kg/day and small / not prominent thymus at 1000 mg/kg/day are indicative of maternal stress at these doses.

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

early or late resorptions

food consumption and compound intake

pre and post implantation loss

total litter losses by resorption

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, the litter data parameters such as the total number of fetuses and weight of male and female fetuses were significantly lower and were considered to be treatment-related developmental toxic effects. At 100 and 300 mg/kg/day the litter parameters such as total number of fetuses, number and weight of live fetuses and sex ratio were statistically comparable to the vehicle control group.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Due to the study design of the OECD 414 body weight change can not be determined in fetals.

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, the litter data parameters such as the total number of fetuses were significantly lower. At 100 and 300 mg/kg/day the litter parameters such as total number of fetuses were statistically comparable to the vehicle control group.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex ratio at at all dose groups was statistically comparable to the vehicle control group.

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day group the litter size were reduced.

Changes in postnatal survival:

not examined

Description (incidence and severity):

Not relevant due to the study design of the OECD 414.

External malformations:

no effects observed

Description (incidence and severity):

There were no signs of external fetal malformations in any of the treatment groups.

Skeletal malformations:

no effects observed

Description (incidence and severity):

There were no signs of teratogenicity / malformations in any of the treatment groups.

Visceral malformations:

no effects observed

Description (incidence and severity):

There were no incidences of major malformations in any of the treatment groups.

Key result

Dose descriptor:

LOAEL

Effect level:

>= 300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

changes in litter size and weights

Remarks on result:

other:

Remarks:

At 1000 mg/kg bw/d final body weight (including uterus) was 26% reduced compared to control group. Significant decreased maternal body weight gains during GD 5-20 at dose groups 300 and 1000 mg/kg/day (-22% and -102%, respectively) as well as weight change corrected for gravid uterine weight at 1000 mg/kg/day was significantly lower (-171%), indicating maternal toxicity. Significantly reduced food consumption at 1000 mg/kg/day group during GD 5 to GD 20 (-39%) and entire gestation period 0 to 20 (-28%) compared to controls indicating maternal toxicity. Due to before mentioned effects the following observations are considered of secondary nature: Decreased gravid uterine weights and significant increase in late resorptions and post implantation loss at 300 and 1000 mg/kg/bw is observed along with significant increase in dams with any resorption and complete resorptions.

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

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

Dose response relationship:

yes

Relevant for humans:

no

TABLE 1.           Details of the Experimental Design, Treatment Schedule, Maternal and Litter Data

Parameters	Group No.	G1	G2	G3	G4
	Dose (mg/kg/day)	0	100	300	1000
Total No. of rats found sperm positive / group		24	24	24	24
Duration of treatment (days)		Gestation Day 5 to 19 (15 days)
No. of rats sacrificed at caesarean section		24	24	24	24
No. of non-pregnant rats at caesarean section		1	0	0	0
No. of pregnant rats at caesarean section		23	24	24	24
No. of litters examined		23	24	22	7
Total no. of foetuses		252	267	203	24
Number of fetuses evaluated
a. External examination		252	267	203	24
b. Visceral examination		126	134	102	12
c. Skeletal examination		126	133	101	12

TABLE 2.           Summary of Clinical Signs, Physical examination and Mortality

Observations	Group No. Dose (mg/kg/day) Total No. of mated rats/group
	G1	G2	G3	G4
	0	100	300	1000
	24	24	24	24
Mortality	None
Clinical signs
Poorly formed faeces containing mostly water	0	0	1	0
Reddish discharge from vagina	0	0	0	16

TABLE 3.           Summary of Maternal Body Weight and Weight Gain

Table 3A: Summary of maternal group mean body weight (g)

Group No.	Dose (mg/kg/day)	No. of Dams	Group mean body weight (g) on day
				0	3	5	8	11	14	17	20
G1	0	23	Mean	212.58	221.07	226.12	231.76	244.28	254.43	277.73	302.53
			SD	14.02	15.38	15.59	15.90	16.87	18.50	20.66	22.59
G2	100	24	Mean	212.41	221.44	226.59	233.31	246.44	256.44	280.57	305.70
			SD	13.80	15.46	14.88	15.39	15.44	15.82	16.68	22.76
G3	300	24	Mean	212.42	221.00	226.92	232.37	243.78	252.23	268.41	286.72
			SD	13.71	15.01	16.51	18.10	17.48	19.25	22.60	33.15
G4	1000	24	Mean	212.38	221.43	225.97	227.89	231.52*	225.69*	228.81*	224.38*
			SD	14.18	16.08	15.77	16.37	20.60	22.79	26.27	29.24

*: Significantly different from vehicle control group

TABLE 3. contd. Summary of Maternal Body Weight and Weight Gain

 

Table 3B: Summary of maternal body weight gain (g)

Period of treatment (days of gestation)	Group No.		G1	G2	G3	G4
	Dose (mg/kg/day)		0	100	300	1000
	No. of Dams		23	24	24	24
0-3		Mean	8.49	9.03	8.59	9.05
		SD	3.02	3.07	2.84	3.33
3-5		Mean	5.05	5.15	5.91	4.54
		SD	1.93	2.20	3.40	1.80
5-8		Mean	5.64	6.72	5.46	1.92*
		SD	2.68	2.34	3.76	3.67
8-11		Mean	12.52	13.13	11.40	3.63*
		SD	2.43	2.21	4.99	8.46
11-14		Mean	10.15	10.00	8.46	-5.83*
		SD	4.27	2.85	4.99	9.31
14-17		Mean	23.30	24.13	16.18*	3.12*
		SD	3.95	4.41	9.64	11.57
17-20		Mean	24.80	25.13	18.31	-4.43*
		SD	4.00	10.04	14.47	11.81

*: Significantly different from vehicle control group

 

TABLE 3. contd. Summary of Maternal Body Weight and Weight Gain

 

Table 3C: Summary of maternal body weight gain (g)

Period of treatment (days of gestation)	Group No.		G1	G2	G3	G4
	Dose (mg/kg/day)		0	100	300	1000
	No. of Dams		23	24	24	24
Pre-treatment period (Days 0-5)		Mean	13.55	14.18	14.50	13.58
		SD	3.98	3.45	4.76	3.88
Treatment Period (Days 5-20)		Mean	76.40	79.11	59.80*	-1.59*
		SD	10.45	13.79	26.12	22.21
Entire gestation period (Days 0-20)		Mean	89.95	93.29	74.30*	12.00*
		SD	12.17	14.97	27.86	23.56

TABLE 3. contd. Summary of Maternal Body Weight and Weight Gain

 

Table3D: Summary of Corrected Body Weight and Body Weight Gain (g)

Group No.	Dose (mg/kg/day)	No. of Dams		Gestation Day 5	Terminal body wt on GD 20	Uterine Wt	Carcass weight (corrected body weight on GD 20)	Corrected Bwt gain.
G1	0	23	Mean	226.12	302.53	60.73	241.80	15.68
			SD	15.59	22.59	11.10	19.28	9.62
G2	100	24	Mean	226.59	305.70	62.17	243.53	16.94
			SD	14.88	22.76	16.71	21.35	14.09
G3	300	24	Mean	226.92	286.72	47.09*	239.63	12.71
			SD	16.51	33.15	21.50	22.88	13.85
G4	1000	24	Mean	225.97	224.38*	9.67*	214.70*	-11.26*
			SD	15.77	29.24	10.09	26.50	19.48

GD: Gestation day;

Corrected body weight on gestation day 20 (Carcass weight) = Terminal body weight on
day 20 - unopened uterine weight.

 

Corrected body weight gain = carcass weight - body weight on day 5.

*: Significantly different from vehicle control group

TABLE 4.           Summary of Food Intake (g/rat /day)

Period of treatment (days of gestation)	Group No.		G1	G2	G3	G4
	Dose (mg/kg/day)		0	100	300	1000
	No. of Dams		23	24	24	24
Intermittent food intake
0-3		Mean	15.77	16.51	16.14	16.16
		SD	2.23	1.62	1.89	1.74
3-5		Mean	16.77	17.54	17.58	16.76
		SD	2.19	1.60	2.85	2.08
5-8		Mean	13.14	13.33	12.78	9.83*
		SD	1.44	1.89	2.45	2.22
8-11		Mean	13.78	14.37	13.20	8.07*
		SD	1.56	1.30	2.16	3.59
11-14		Mean	14.68	14.84	14.49	7.68*
		SD	1.49	1.37	2.79	4.21
14-17		Mean	15.79	16.41	15.05	10.96*
		SD	1.68	1.35	3.78	4.11
17-20		Mean	14.13	15.03	13.77	9.39*
		SD	2.08	3.89	4.78	4.23

TABLE 4 contd. Summary of Food Intake (g/rat /day)

Group No.		G1	G2	G3	G4
Period of treatment Dose (mg/kg/day)		0	100	300	1000
No. of Dams		23	24	24	24
Pre-treatment period (Days 0-5)	Mean	16.17	16.92	16.72	16.40
	SD	1.52	1.53	2.14	1.81
Treatment Period (Days 5-20)	Mean	14.31	14.80	13.86	9.19*
	SD	1.31	1.59	2.78	2.60
Entire gestation period (Days 0-20)	Mean	14.77	15.33	14.57	10.99*
	SD	1.26	1.49	2.47	2.13

*: Significantly different from vehicle control group

TABLE 5.           Summary of Maternal Data

	Group No.	G1	G2	G3	G4
Parameters	Dose (mg/kg/day)	0	100	300	1000
	No. of Dams	23^#	24	24	24
Gravid uterine weight (g)	Mean	60.73	62.17	47.09*	9.67*
	SD	11.10	16.71	21.50	10.09
Number of Corpora lutea	Total	303	329	325	319
	Mean	13.17	13.71	13.54	13.29
	SD	2.15	1.68	1.53	1.92
Number of Implantations	Total	264	279	277	270
	Mean	11.48	11.63	11.54	11.25
	SD	2.54	2.93	2.26	2.40
Early Resorptions	Total	9	10	30	21
	Mean	0.39	0.42	1.25	0.88
	SD	0.89	0.78	2.44	1.26
Late Resorptions	Total	3	2	44	225
	Mean	0.13	0.08	1.83*	9.38*
	SD	0.63	0.28	3.58	2.87
Pre-implantation Loss	Total number	39	50	48	49
	Mean	1.70	2.08	2.00	2.04
	SD	1.46	2.52	2.06	1.90
Post-implantation Loss	Total number	12	12	74	246
	Mean	0.52	0.50	3.08*	10.25*
	SD	7.70	9.50	30.79	15.55
Dams with any Resorption	Total	7	9	19*	7
Dams with all Resorption	Total	0	0	2	17*

*: Significantly different from vehicle control group;^#: one animal not pregnant;

All mean values were calculated with numbers of dams mentioned in table

TABLE 5. contd. Summary of Maternal Data (% per Litter)

	Group No.	G1	G2	G3	G4
Parameters	Dose (mg/kg/day)	0	100	300	1000
	No. of Dams	23	24	24	24
Early Resorptions	Mean	2.94	4.78	10.79	7.89
	SD	6.22	9.59	20.31	11.75
Late Resorptions	Mean	1.09	0.62	15.89	83.92
	SD	5.21	2.10	28.05	19.39
Pre-implantation Loss	Mean	13.27	15.30	14.59	15.15
	SD	12.61	18.41	14.67	14.15
Post-implantation Loss	Mean	4.02	5.39	26.67	91.81
	SD	7.70	9.50	30.79	15.55
Implantation Index	Mean	86.73	84.70	85.41	84.85
	SD	12.61	18.41	14.67	14.15

Note: Values are calculated according Indices (see above "Examination", subpoint "indices") and not rounded.

TABLE 6.           Summary of Litter Data

	Group No.	G1	G2	G3	G4
Parameters	Dose (mg/kg/day)	0	100	300	1000
	No. of Dams	23	24	24	24
No. of litters		23	24	22	7
Total no. of fetuses		252	267	203	24*
Mean litter size		11.0	11.1	9.2	3.4
Dead fetuses	Total	0	0	0	0
	%	0	0	0	0
Total live fetuses
a.  Number		252	267	203	24*
	%	100	100	100	100
b.  Weight (g)	Mean	3.54	3.59	3.38	2.60*
	SD	0.25	0.24	0.36	0.40
Live male fetuses
a.  Number		127	136	97	9*
	%	50	51	48	38
b.  Weight (g)	Mean	3.65	3.67	3.50	2.59*
	SD	0.29	0.25	0.35	0.52
Live female fetuses
a.  Number		125	131	106	15*
	%	50	49	52	62
b.  Weight (g)	Mean	3.40	3.49	3.29	2.78*
	SD	0.25	0.26	0.38	0.23
Sex Ratio - Male : Female		1:0.98	1:0.96	1:1.09	1:1.67

*: Significantly different from vehicle control group

TABLE 7.           Summary of Gross Pathological Findings

Group No.	G1	G2	G3	G4
Parameters Dose (mg/kg/day)	0	100	300	1000
1.  No. of rats subjected to caesarean section	24	24	24	24
2.  No. of rats pregnant at caesarean section	23	24	24	24
3.  No. of rats showing gross pathology
Adrenal enlarged – Bilateral	0	0	1	12
Thymus: Small	0	0	0	10
Thymus: Not prominent	0	0	0	9

Fetal External Observation (see Table 8 below)

At 1000 mg/kg/day anasarca was found in three foetuses which was considered as secondary to the maternal toxicity.

At 300 mg/kg/day anasarca was found in 4 fetuses of one dam (Rs4625). This dam exhibited remarkable findings prior to treatment, such as reduction in body weight, body weight gain and food intake (Appendix 3 and 4) indicating a bad health condition from begin on. The bad health condition of this dam is seen in smaller body weight gain and poor food intake during gestation days 0-3 and 3-5 (time prior to treatment) compared to other females of this dosing group. At termination the corrected body weight and corrected body weight gain was clearly decreased compared to all other dams of this dose group. Further,  poorly formed faeces containing mostly water was described on GD 20 for this dam. No abnormality were detected at clinical and physical examination at any other dam of this dosing group. Thus, the appearance of anasarca in fetals of this dam (Rs4625) of the 300 mg/kg/day group is considered as secondary to maternal toxicity. There were no incidences of major external malformations in any of the treatment groups.

Table 8.                    Summary of Fetal external Observations

Group No.	G1			G2			G3			G4
Dose (mg/kg/day)	0			100			300			1000
No. of litters examined	23			24			22			7
No. of fetuses examined	252			267			203			24
	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %
Normal Variant	None
Minor Anomalies
Anasarca	0	0.00	0.00	0	0.00	0.00	4*	1.97	3.00	3*	12.50*	17.86*
Major Malformations	None

*: Significantly different from vehicle control group

 

 

Fetal Visceral Observation (See Table 9 below)

There was an incidence of a fetus with slight dilatation of renal pelvis of kidney in a fetus from control (1/126) which is indicated as anormal as well as a minor variant. There were no observations in any of the other treated groups and there were no incidences of major malformations in any of the treatment groups.

Table 9.        Summary of Fetal Visceral Observations (incidence and percentage)

Group No.	G1			G2			G3			G4
Dose (mg/kg/day)	0			100			300			1000
No. of litters examined	23			24			22			7
No. of fetuses examined	126			134			102			12
	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %
Normal Variant
Kidney renal pelvis dila. (Rt/Lt/B) (slight)	1	0.79	1.00	1	0.75	1.00	0	0.00	0.00	0	0.00	0.00
Minor Anomalies
Kidney renal pelvis dila. (Rt/Lt/B) (moderate)	1	0.79	1.00	0	0.00	0.00	0	0.00	0.00	0	0.00	0.00
Major Malformations	None

 

Fetal Skeletal Observation (see Table 10 below)

The incidences/percentage of normal variants [delayed skeletal ossification (DSO) and incomplete/poor ossification (INO/PO)] and minor anomalies were comparable between the vehicle control and 100 mg/kg/day dose.

The statistically significant changes in normal variants [delayed skeletal ossification (DSO) and incomplete/poor ossification (INO/PO)] and minor anomalies noted at 300 and 1000 mg/kg/day are expected changes mainly due to the poor development of the fetuses at these doses. There were major malformations in any of the treatment groups.

 

Table 10.       Summary of Fetal Skeletal Observations (incidence and percentage)

Group No.	G1			G2			G3			G4
Dose (mg/kg/day)	0			100			300			1000
No. of litters examined	23			24			22			7
No. of fetuses examined	126			133			101			12
	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %
Normal Variant
DSO:  CV:centra:7/7	6	4.76	5.04	3	2.26	3.27	16*	15.84*	18.69*	9*	75.00*	82.14
TV:centra:1/13	1	0.79	0.87	0	0.00	0.00	2	1.98	3.03	2*	16.67*	28.57
SV:centra:1/4	0	0.00	0.00	0	0.00	0.00	1	0.99	1.52	0	0.00	0.00
CdV:centra:1/4	11	8.73	8.54	31*	23.31*	22.61*	32*	31.68*	30.94*	3	25.00*	25.00
CdV:centra:2/4	3	2.38	2.46	8	6.02	6.88	4	3.96	5.19	2*	16.67*	7.14*
CdV:centra:3/4	0	0.00	0.00	0	0.00	0.00	0	0.00	0.00	2*	16.67*	28.57*
CdV:centra:4/4	0	0.00	0.00	0	0.00	0.00	3	2.97	4.55*	0	0.00	0.00
CdV:arch:1/2	0	0.00	0.00	7*	5.26*	6.05*	2	1.98	2.16	2*	16.67*	28.57*
CdV:arch:2/2	0	0.00	0.00	0	0.00	0.00	1	0.99	1.52	0	0.00	0.00
F limb:Metacarp.:1/4	46	36.51	38.24	63	47.37	47.25	64*	63.37*	63.65	11*	91.67*	92.86

*: Significantly different from vehicle control group

 

 

Table 10.        contd. Summary of Fetal Skeletal Observations(incidence and percentage)

Group No.	G1			G2			G3			G4
Dose (mg/kg/day)	0			100			300			1000
No. of litters examined	23			24			22			7
No. of fetuses examined	126			133			101			12
	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %
Normal Variant
DSO:  F limb:Pr. Phal:2/2	118	93.65	93.91	131*	98.50	98.61	101*	100.00*	100.00	12	100.00*	100.00
F limb:Dt. Phal:1/4	1	0.79	0.87	8*	6.02*	7.09*	6*	5.94*	6.26*	0	0.00	0.00
F limb:Dt. Phal:4/4	2	1.59	1.96	5	3.76	6.29	19*	18.81*	22.62*	5*	41.67*	39.29
H limb:Metatar.:1/4	2	1.59	1.74	0	0.00	0.00	3	2.97	4.55	1	8.33*	14.29*
H limb:Pr. Phal:1/4	0	0.00	0.00	1	0.75	2.08	0	0.00	0.00	0	0.00	0.00
H limb:Dt. Phal:1/5	1	0.79	0.87	1	0.75	0.69	0	0.00	0.00	0	0.00	0.00
H limb:Dt. Phal:5/5	3	2.38	2.83	7	5.26	8.03	25*	24.75*	28.65*	6*	50.00*	42.86
INO/PO: Skull bones	0	0.00	0.00	0	0.00	0.00	3	2.97	4.55*	5*	41.67*	57.14
Parietal	0	0.00	0.00	1	0.75	0.69	1	0.99	0.91	0	0.00	0.00
Pa, Ipa	1	0.79	0.87	7*	5.26	6.91*	11*	10.89*	11.98*	1*	8.33*	3.57*

*: Significantly different from vehicle control group

 

  Table 10.        contd. Summary of Fetal Skeletal Observations(incidence and percentage) 

Group No.	G1			G2			G3			G4
Dose (mg/kg/day)	0			100			300			1000
No. of litters examined	23			24			22			7
No. of fetuses examined	126			133			101			12
	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %
Normal Variant
INO/PO: Inter pariet.	0	0.00	0.00	1	0.75	1.04	1	0.99	0.91	0	0.00	0.00
Fr, Pa, Ipa	1	0.79	0.72	2	1.50	2.68	3	2.97	3.94	6*	50.00*	39.29
Supraocci	1	0.79	0.72	2	1.50	2.78	2	1.98	2.42	2*	16.67*	17.86*
Stern:# 1-4	1	0.79	0.72	1	0.75	1.04	2	1.98	3.03	0	0.00	0.00
Stern:# 1	1	0.79	0.87	3	2.26	3.37	13*	12.87*	14.18*	2*	16.67*	21.43*
Stern:# 2	14	11.11	10.37	24	18.05	17.82*	56*	55.45*	54.48	1	8.33	7.14*
Stern:# 3	1	0.79	0.87	2	1.50	2.78	8*	7.92*	9.44*	3*	25.00*	21.43*
Stern:# 4	2	1.59	1.74	3	2.26	3.47	14*	13.86*	15.19*	3*	25.00*	21.43*
Stern:# 5	50	39.68	41.05	52	39.10	37.55	62*	61.39*	57.67	1*	8.33*	7.14
Stern:# 6	34	26.98	24.46	35	26.32	27.68	27	26.73	25.93	0*	0.00*	0.00*

*: Significantly different from vehicle control group

 

 

Table 10.        contd. Summary of Fetal Skeletal Observations(incidence and percentage)

Group No.	G1			G2			G3			G4
Dose (mg/kg/day)	0			100			300			1000
No. of litters examined	23			24			22			7
No. of fetuses examined	126			133			101			12
	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %
Normal Variant
INO/PO: CV:centra:1/7	3	2.38	2.43	9	6.77	6.03	13*	12.87*	10.63*	1	8.33	7.14*
CV:centra:2/7	9	7.14	6.68	7	5.26	5.01	8	7.92	13.18*	0	0.00*	0.00
CV:centra:3/7	7	5.56	5.33	4	3.01	2.64	7	6.93	6.41	1	8.33	7.14*
CV:centra:4/7	2	1.59	1.35	0	0.00	0.00	5	4.95	4.59	1	8.33*	3.57*
CV:centra:5/7	3	2.38	2.22	4	3.01	2.72	0	0.00	0.00	0	0.00	0.00
CV:centra:6/7	2	1.59	1.24	3	2.26	1.98	8*	7.92*	6.86*	0	0.00	0.00
CV:centra:7/7	1	0.79	0.72	0	0.00	0.00	0	0.00	0.00	0	0.00	0.00
TV:centra:1/13	0	0.00	0.00	0	0.00	0.00	1	0.99	1.52	3*	25.00*	17.86*
TV:centra:2/13	0	0.00	0.00	0	0.00	0.00	1	0.99	1.52	0	0.00	0.00
SV:centra:2/4	0	0.00	0.00	0	0.00	0.00	1	0.99	1.52	0	0.00	0.00

*: Significantly different from vehicle control group

 

  

Table 10.        contd. Summary of Fetal Skeletal Observations(incidence and percentage)

Group No.	G1			G2			G3			G4
Dose (mg/kg/day)	0			100			300			1000
No. of litters examined	23			24			22			7
No. of fetuses examined	126			133			101			12
	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %
Normal Variant
INO/PO:   SV:arch:2/4	0	0.00	0.00	0	0.00	0.00	1	0.99	1.52	0	0.00	0.00
Ileum	0	0.00	0.00	0	0.00	0.00	3	2.97	4.55*	0	0.00	0.00
Pubis	2	1.59	1.59	0	0.00	0.00	5	4.95	7.58*	5*	41.67*	50.00
F limb:Metacarp.:1/4	0	0.00	0.00	0	0.00	0.00	1	0.99	0.65	0	0.00	0.00
SV:centra&arch:3,4	0	0.00	0.00	0	0.00	0.00	0	0.00	0.00	1*	8.33*	14.29*
SV:centra:3,4	0	0.00	0.00	0	0.00	0.00	0	0.00	0.00	1*	8.33*	14.29*
SV:arch:3,4	0	0.00	0.00	0	0.00	0.00	0	0.00	0.00	1*	8.33*	14.29*

*: Significantly different from vehicle control group

 

Table 10.        contd. Summary of Fetal Skeletal Observations(incidence and percentage)

Group No.	G1			G2			G3			G4
Dose (mg/kg/day)	0			100			300			1000
No. of litters examined	23			24			22			7
No. of fetuses examined	126			133			101			12
	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %
Minor Anomalies
HYPOPLASTIC:Stern:# 1	1	0.79	0.72	1	0.75	1.04	6*	5.94*	7.62*	1*	8.33*	7.14*
Stern:# 2	1	0.79	0.72	3	2.26	3.82	18*	17.82*	16.94*	1*	8.33*	3.57*
Stern:# 3	1	0.79	0.72	1	0.75	1.04	5	4.95	6.71*	0	0.00	0.00
Stern:# 4	1	0.79	0.72	1	0.75	1.04	6*	5.94*	7.62*	0	0.00	0.00
Stern:# 5	17	13.49	13.23	18	13.53	14.10	20	19.80	17.94	1	8.33	3.57
Stern:# 6	2	1.59	1.24	1	0.75	0.83	3	2.97	6.36*	0	0.00	0.00
TV:centra:1/13	0	0.00	0.00	0	0.00	0.00	2	1.98	3.03	1*	8.33*	14.29*
DB: TV:centra:1/13	8	6.35	7.97	6	4.51	7.29	20*	19.80*	21.45*	5*	41.67*	42.86
TV:centra:2/13	1	0.79	1.09	2	1.50	1.19	9*	8.91*	9.03*	2*	16.67*	21.43*
TV:centra:3/13	0	0.00	0.00	1	0.75	0.69	0	0.00	0.00	0	0.00	0.00

  *: Significantly different from vehicle control group

 

 

Table 10.        contd. Summary of Fetal Skeletal Observations(incidence and percentage)

Group No.	G1			G2			G3			G4
Dose (mg/kg/day)	0			100			300			1000
No. of litters examined	23			24			22			7
No. of fetuses examined	126			133			101			12
	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %
Minor Anomalies
DB: TV:centra:6/13	0	0.00	0.00	0	0.00	0.00	0	0.00	0.00	1*	8.33*	3.57*
:LV:centra:1/6	0	0.00	0.00	0	0.00	0.00	1	0.99	0.91	0	0.00	0.00
ASY OSSI:  stern:# 1	0	0.00	0.00	0	0.00	0.00	3	2.97	3.94*	3*	25.00*	25.00*
stern:# 3	0	0.00	0.00	0	0.00	0.00	2	1.98	2.42	3*	25.00*	25.00*
stern:# 4	1	0.79	0.87	0	0.00	0.00	2	1.98	2.42	4*	33.33*	28.57*
stern:# 3,4	0	0.00	0.00	4*	3.01	2.33	2	1.98	2.65	0	0.00	0.00
LV:centra:2/6	0	0.00	0.00	0	0.00	0.00	0	0.00	0.00	1*	8.33*	14.29*
ASY DB:TV:centra:1/13	0	0.00	0.00	1	0.75	2.08	1	0.99	0.65	0	0.00	0.00
SPLIT:  Stern:# 1	0	0.00	0.00	0	0.00	0.00	1	0.99	1.52	10*	83.33*	85.71
Stern:# 2	0	0.00	0.00	0	0.00	0.00	0	0.00	0.00	1*	8.33*	3.57*

*: Significantly different from vehicle control group

 

  

Table 10.        contd. Summary of Fetal Skeletal Observations(incidence and percentage)

Group No.	G1			G2			G3			G4
Dose (mg/kg/day)	0			100			300			1000
No. of litters examined	23			24			22			7
No. of fetuses examined	126			133			101			12
	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %
Minor Anomalies
SPLIT:  Stern:# 3	0	0.00	0.00	0	0.00	0.00	2	1.98	3.03	10*	83.33*	78.57
Stern:# 4	0	0.00	0.00	0	0.00	0.00	2	1.98	3.03	10*	83.33*	78.57
Stern:# 5	0	0.00	0.00	0	0.00	0.00	0	0.00	0.00	1*	8.33*	3.57*
Stern:1-4	0	0.00	0.00	0	0.00	0.00	1	0.99	1.52	0	0.00	0.00
Stern:1,3,4	0	0.00	0.00	0	0.00	0.00	4*	3.96*	6.06*	0	0.00	0.00
TV:centra:1/13	0	0.00	0.00	0	0.00	0.00	1	0.99	1.52	1*	8.33*	7.14*
EXTRA:LV:centra,arch:# 7	0	0.00	0.00	0	0.00	0.00	1	0.99	1.52	0	0.00	0.00
RIB(Rt/Lt/B):# 14	0	0.00	0.00	2	1.50	1.39	1	0.99	1.52	0	0.00	0.00
ACCESSORY: RIB(Rt/Lt/B):# 14	4	3.17	3.20	5	3.76	3.61	6	5.94	7.12	1	8.33	14.29*
RUDIMENTARY: RIB(Rt/Lt/B):# 14	44	34.92	34.63	60	45.11	49.36	59*	58.42*	55.02	8*	66.67*	67.86

*: Significantly different from vehicle control group

 

  

Table 10.        contd. Summary of Fetal Skeletal Observations(incidence and percentage)

Group No.	G1			G2			G3			G4
Dose (mg/kg/day)	0			100			300			1000
No. of litters examined	23			24			22			7
No. of fetuses examined	126			133			101			12
	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %	Inc.	Fetus %	Litter %
Minor Anomalies
UNILAT OSSI:TV:centra:1/13	0	0.00	0.00	0	0.00	0.00	1	0.99	1.52	1*	8.33*	14.29*
F.limb(Rt/Lt/B(+/++)) flexed at wrist	0	0.00	0.00	0	0.00	0.00	1	0.99	0.65	0	0.00	0.00
WAVY:RIB(Rt/Lt/B)(+/++):3/13	0	0.00	0.00	1	0.75	2.08	0	0.00	0.00	0	0.00	0.00
Major Malformations	None

*: Significantly different from vehicle control group

Conclusions:

Based on the above findings, it is concluded that the No Observed Adverse Effect Level (NOAEL) for Maternal toxicity and Fetal developmental toxicity is 100 mg/kg/day in Wistar rats when Hostanox O 3 P was administered orally by gavage during GD 5-19 under the test conditions and doses employed in this study due to:
Treatment related clinical sign of reddish vaginal discharge at 1000 mg/kg /day, significant reduction in maternal body weight gains at 300 and 1000 mg/kg/day and food consumption at 1000 mg/kg/day. Due to the before mentioned effects the following observations are considered of secondary nature: Decreased gravid uterine weights and significant increase in late resorptions and post implantation loss at 300 and 1000 mg/kg/day along with significant increase in dams with any resorption and complete resorption.

Executive summary:

The objective of this study was to evaluate the embryo-fetal developmental toxicity of test item Hostanox O 3 P when administered to pregnant Wistar rats by oral route during gestation days (GD) 5 to GD19.

Ninety six presumed pregnant Wistar rats were assigned to four groups by body weight stratification (Group 1 to Group 4) and each group (G1: control, G2: low dose, G3: mid dose and G4: high dose) consisted of 24 presumed pregnant rats (gestation day 0). Day `0' of gestation for each individual female rat in the study was considered as the day on which vaginal smear was found positive for sperm.

The test item, Hostanox O 3 P was suspended in sunflower oil and administered orally (by gavage) to presumed pregnant rats once daily from GD 5 to 19 at the dose levels of 100, 300 and 1000 mg/kg/day for low (G2), mid (G3) and high (G4) dose group rats, respectively. Rats in the vehicle control (G1) group received the vehicle (sunflower oil) alone. A constant dose volume of 10 mL/kg body weight was administered to all groups. The mated females were observed twice daily for clinical signs (during treatment period), mortality and morbidity. Body weights were recorded on GD0, 3, 5, 8, 11, 14, 17 and 20. Food consumption was determined on GD 3, 5, 8, 11, 14 and 17. The food left over was also recorded on Day 20 of presumed gestation.

Caesarean section was performed for all rats on GD 20 and dams were examined for gross pathological changes. The uterus from all the animal were removed (by laparotomy) and the contents were examined.The uteri were weighed and examined for the number of implantation sites, early and late resorptions, and number of live and dead fetuses. The number of corpora lutea was counted on each ovary. All the fetuses were sexed, weighed and examined for external malformations.Approximately half the number of fetuses from each dam was examined for visceral malformations and the remaining half was evaluated for skeletal malformations.

·      There was no mortality at any of the doses tested.

·      At 1000 mg/kg/day, treatment-related clinical sign of reddish discharge from the vagina was observed between GD 13 - 20. This finding was associated with dams with resorptions at necropsy.

- At terminal sacrifice, there were gross pathological changes of enlarged adrenals in dams treated at 300 (1/24) and 1000 mg/kg/day (10/24) indicative of maternal stress at these doses. In addition, gross finding of small /not prominent thymus at 1000 mg/kg/day dose level was also indicative of maternal stress at this dose.

·     At 1000 mg/kg/day final body weight (including uterus) was 26 % reduced compared to control group. At 300 and 1000 mg/kg/day, there were treatment-related significant reduction in maternal body weight gains during the treatment period GD5 to 20 (-22% and -102%, respectively) and entire gestation period 0 to 20 (-17% and -87%, respectively). The body weight change corrected for gravid uterine weight at 1000 mg/kg/day was significantly lower (-172%) indicating maternal toxicity

·      At 1000 mg/kg/day, the food consumption during the treatment period GD5 to 20 (-39%) and entire gestation period 0 to 20 (-28%) was significantly lower as compared to vehicle control group indicating maternal toxicity.

·      At 300 and 1000 mg/kg/day, the gravid uterine weights were significantly lower (-17% and -45%, respectively) and there was significant increase in late resorptions and post implantation loss. There was significant increase in dams with any resorption and complete resorptions at 300 and 1000 mg/kg/day, respectively indicating developmental toxicity. Gross evaluation of placenta revealed no findings.

·      At 1000 mg/kg/day, the litter data parameters such as the total number of fetuses and weight of male and female fetuses were significantly lower and were consequently considered to be treatment-related developmental toxic effects.

·      Fetal visceral observations revealed no signs of fetal malformations or developmental toxicity at the doses tested.

·      Fetal skeletal examination revealed no signs of teratogenicity / malformations in the tested dose levels. The statistically significant increase in the incidence and percentage of normal variants and minor anomalies in some of the bone components at 300 and 1000 mg/kg/day doses are expected changes mainly due to the poor development of the fetuses at these doses.

Based on the above findings, it is concluded that the No Observed Adverse Effect Level (NOAEL) for Maternal toxicity and Fetal developmental toxicity is 100 mg/kg/day in Wistar rats when Hostanox O 3 P was administered orally by gavage during GD 5 to 19 under the test conditions and doses employed in this study due to:

Treatment related clinical sign of reddish vaginal discharge at 1000 mg/kg /day, significant reduction in maternal body weight gains at 300 and 1000 mg/kg/day and food consumption at 1000 mg/kg/day. Due to the before mentioned effects the following observations are considered of secundary nature: Decreased gravid uterine weights and significant increase in late resorptions and post implantation loss at 300 and 1000 mg/kg/day along with significant increase in dams with any resorption and complete resorption.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

March - August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

equivalent or similar to guideline

Guideline:

other: combination of OECD 414 and OECD 407

Principles of method if other than guideline:

The objective of this study was to determine the toxicity potential of the test item Hostanox O 3 P when administered repeatedly by oral route for 14 consecutive days for males and non-pregnant female Wistar rats. Satellite group rats were treated for 2 days and 9 days at the same doses tested for repeat dose phase groups. In addition Embryo-Fetal Developmental (EFD) toxicity was evaluated in pregnant Wistar rats which were exposed to Hostanox O 3 P by oral route during gestation days GD 5 to GD 19.

GLP compliance:

no

Remarks:

study performed in a GLP certified laboratory

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Vivo Bio Tech Ltd, Sy # 349/A, Pregnapur-502311, Gajwel Mandal, Medak District, Telangana
- Age at study initiation: 15 to 16 weeks
- Weight at study initiation:
Repeat Dose Phase
Males Females
G1: 365.62 ± 34.22 210.86 ± 10.10
G2: 362.90 ± 29.71 208.96 ± 5.91
G3: 361.69 ± 30.03 208.36 ± 7.17
G4: 361.69 ± 28.36 213.45 ± 11.53

Satellite Groups
Males Females
G1S: 364.41 ± 22.67 215.95 ± 12.26
G2S: 362.54 ± 26.84 213.78 ± 9.88
G3S: 363.08 ± 25.41 223.96 ± 10.10
G4S: 366.98 ± 25.39 216.20 ± 11.65

Embryo Fetal Developmental (EFD) Phase
Females
G1EFD: 224.44 ± 10.24
G2EFD: 225.20 ± 5.61
G3EFD: 225.85 ± 5.53
G4EFD: 225.10 ± 7.96
- Housing:
Maximum of two rats per cage were housed in standard polysulfone cages (size: approximately Length 425 x Breadth 266 x Height 185 mm), with stainless steel top grill having facilities for pelletted food and polycarbonate drinking water bottle with stainless steel sipper tubes. Additionally, polycarbonate rat huts were provided to the animals as an environmental enrichment objects in the cages that either provide shelter or exercising opportunities to minimize animal stress and promote overall well-being.

For the EFD Phase, the following was the housing pattern during different periods of the experiment:

i. Acclimatization period: Two rats per cage were housed.
ii. Mating period: Female rats were cohabited with males in 1:1 ratio.
iii. Post-mating / Gestation period: After confirming mating, females were housed individually. Additionally, polycarbonate rat huts was provided to the animals as an environmental enrichment objects in the cages that either provide shelter or exercising opportunities to minimize animal stress and promote overall well-being.

- Diet (e.g. ad libitum):
Teklad Certified (2014C) Global 14 % Protein Rodent Maintenance Diet - Pellet (Certified) manufactured by Harlan Laboratories, P.O.Box 44220, Madison Wi 53744-4220, was provided ad libitum to the rats.

- Water (e.g. ad libitum):
Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd., Mumbai - 400 001, India was provided ad libitum to rats in polycarbonate bottles with stainless steel sipper tubes.

- Acclimation period: min 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 66 – 68 %
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
From: 24.03. To: 20.04.2017

Route of administration:

oral: gavage

Vehicle:

other: sunflower oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility
- Concentration in vehicle: 10. 30 or 60 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw/d
- Lot/batch no. (if required): T051

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulations were analyzed for active ingredient concentration (a.i.) and homogeneity in duplicate sets a day prior to the initiation of treatment and 3 days prior to the termination of treatment period.

The prepared formulations were sampled in duplicate sets wherein one set was used for analysis and another was kept as back up set which was stored in the experimental room. For each set, duplicate samples were drawn from top, middle and bottom layers of each dose formulation. In case of control, duplicate samples from middle layer were drawn. Dose formulations were sent to Analytical R&D Department of Advinus Therapeutics Limited for formulation analysis. Dose formulation samples were analyzed for Hostanox O 3 P content as per the Analytical method validated under Advinus Study No. G11434.

Formulations were considered acceptable when the overall mean result (calculated using all the 6 replicate values) of all the layers and mean of each layers was within ± 15.0 % of the claimed concentration and the relative standard deviation (% RSD, calculated using all the 6 replicate values) of assay of top, middle and bottom layers was equal to or less than 10.0 %.

Details on mating procedure:

For the EFD Phase:
During the mating period, female rats were cohabited with males in 1:2 ratio. When sperm was detected in the vaginal smear or if a vaginal plug was observed in the morning, the animal was considered to be mated. This day was considered as day 0 of gestation.
The mated female rats obtained each day were assigned to the treatment groups and vehicle control groups by body weight stratification. This procedure was continued till there were sufficient numbers of Day 0 mated rats for the study.
The mated female rats obtained each day were assigned to the treatment groups and vehicle control groups by body weight stratification. This procedure was continued till there were sufficient numbers of Day 0 mated rats for the study.

Duration of treatment / exposure:

Repeated dose rats: 14 days
Satellite groups: 2 or 9 days (necropsy on days 3 and 10, respectively)
Embryo-Fetal Develpomental groups: gestation days 5 to 19

Frequency of treatment:

once daily

Duration of test:

refer to "duration of treatment"

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

The repeat dose phase and the statellite group consisted of 6 rats/sex/group,
EFD phase consisted of 6 presumed pregnant rats (gestation day 0)

Control animals:

yes, concurrent vehicle

Details on study design:

The test item, Hostanox O 3 P was suspended in vehicle [sunflower oil] and administered to Wistar rats orally (by gavage) at the doses of 100 (low dose), 300 (Mid dose) and 600 mg/kg/day (high dose) at an equivolume of 10 mL/kg/day to 3 cohorts of rats, the repeat dose phase (G2 to G4), the statellite group (G2S to G4S) and the Embryo Fetal Developmental (EFD) phase (G2EFD to G4EFD). The repeat dose phase and the statellite group consisted of 6 rats/sex/group, while the EFD phase consisted of 6 presumed pregnant rats (gestation day 0). The rats in vehicle control groups (G1/G1S/G1EFD) received vehicle alone at an equivolume of 10 mL/kg/day.
The test item was administered to the repeat dose phase rats once daily for 14 days, for the statellite group rats once daily for 2 and 9 days and for presumed pregnant EFD phase rats once daily from GD 5 to GD 19.

- Parameters analysed / observed:
Assessments included clinical signs, body weights, food consumption, clinical laboratory investigations of blood (for hematology, acute phase proteins, clinical chemistry and hormone analysis), gross pathology, and histopathological evaluation (repeat dose phase and statellite group). The microscopic examination was initially performed for the control and high dose groups of males and females, and extended to the following organs of lower dose groups as higher incidence of lesions was present in high dose groups:
Satellite groups (Day 3 sacrifice): Liver, duodenum, jejunum and adrenals
Satellite groups (Day 10 sacrifice): Liver, duodenum, jejunum, ileum with Peyer’s patch, spleen and adrenals
Repeat dose groups: Liver, duodenum, jejunum, ileum with Peyer’s patch, spleen, thymus and adrenals

For the EFD phase rats, caesarean section was performed for all rats on GD 20 and dams were examined for gross pathological changes. The uterus from all the animals were removed (by laparotomy) and the contents were examined. The uteri were weighed and examined for the number of implantation sites, early and late resorptions, and number of live and dead fetuses. The number of corpora lutea was counted on each ovary. All the fetuses were sexed, weighed and examined for external malformations. Approximately half the number of fetuses from each dam was examined for visceral malformations.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
Repeated dose phase:
- Individual body weight was recorded on Days 1, 4, 8, 11 and 14.
Satelite groups:
- Individual body weight was recorded on Days 1, 4 and 9
EFD Phase_
- All females included in the study were weighed on gestation days 0, 3, 5, 8, 11, 14, 17 and 20

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Repeated dose phase
- Food intake was measured during days 1-4, 4-8, 8-11 and 11-14.
Satelite groups:
- Food intake was measured during days 1-2 or 1-4, 4-9
EFD phase:
- About 200 g (food input) was provided on Day 0. The food output was recorded and replenished to about 200 g on Days 3, 5, 8, 11, 14 and 17 and food output on Day 20 of presumed gestation was recorded.
Food intake was expressed as grams of food consumed daily per rat. Intermittent food intake i.e., 0-3, 3-5, 5-8, 8-11, 11-14, 14-17 and 17-20 was calculated. Further food intake for pre-treatment (0-5), treatment (5-20) and for entire gestation period (0-20) was derived and presented only for rats pregnant at caesarean section

POST MORTEM OBSERVATION:
EDF phase:
-The uterus from all the animals were removed (by laparotomy) and the contents were examined. The uteri were weighed and examined for the number of implantation sites, early and late resorptions, and number of live and dead fetuses. The number of corpora lutea was counted on each ovary.

OTHER:
Clinical laboratory investigations of blood (for hematology, acute phase proteins, clinical chemistry and hormone analysis), gross pathology, and histopathological evaluation (repeat dose phase and statellite group).

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
- Other:

Fetal examinations:

All the fetuses were sexed, weighed and examined for external malformations. Approximately half the number of fetuses from each dam was examined for visceral malformations.

Statistics:

The statistical analysis was carried out using the validated package in Excel and also using licensed copies of SYSTAT Statistical package ver.12.0.

The data on body weight, body weight change, gravid uterine weight, body weight change corrected to gravid uterine weight, food consumption, hormonal analysis data were analyzed using Analysis of Variance (ANOVA) after testing for homogeneity for intra group variance using Levene’s test. Where intra group variances are heterogeneous, ANOVA was performed after suitable transformation of data. Comparison of means between treatment groups and control group was done using Dunnett’s test if the overall treatment, ‘F’ test is found to be significant.

Fetal weight for male and female were analyzed using Analysis of Convariance (ANCOVA) taking litter size as covariate for group.

Number of corpora lutea, number of implantations, early and late resorptions, pre-implantation and post-implantation loss, external observations for variations were analyzed using Kruskal Wallis test for group comparison. Mann-Whitney pairwise comparison of the treated groups with the control group was performed, when the group differences are significant.

The incidence of with and without resorptions in dams were tested using Cochran Armitage trend test followed by Fisher’s exact test for group association.

Statistically significant differences (p<0.05), indicated by the aforementioned tests are designated (*) throughout the report.

Data captured using ProvantisTM: Parameters such as clinical pathology data - hematology and clinical chemistry (other than acute phase proteins and hormones) were analysed using ProvantisTM built-in statistical tests

Indices:

n.a.

Historical control data:

n.a.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 600 mg /kg/day reddish discharge from vagina was observed in 3/ 6 dams between GD 16 to 20.

Dermal irritation (if dermal study):

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights of 600 mg/kg/day dams was significantly lower on GD 20 compared to vehicle control

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In high dose dams the maternal food intake was significantly lower (-29%) as compared to vehicle control group

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The neutrophil, monocyte counts and total leukocyte counts were higher at 600 mg/kg dose group on gestation Day 20.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Increased ALT activity in ≥300 mg/kg dose groups and decreased ALP activity at 600 mg/kg.
Increased total bilirubin level in all treated groups. The direct and indirect bilirubin levels were higher in ≥300 mg/kg dose groups.
Triglyceride level was lower at all treated groups.
All the above indicated changes were consistent across different intervals with minimal variation and were associated with the morphological changes in liver (cytoplasmic rarefaction/vacuolation in hepatocytes) and intestinal segments (lymphangiectasis).

Acute Phase Proteins (haptoglobin and A1 alpha glycoprotein):
Increased haptoglobin in all treated groups and increased A1 alpha glycoprotein at 600 mg/kg.

Hormone analysis:
There were no test item-related changes in the hormone parameters. The hormone analysis data from satellite, repeat and EFD groups did not reveal any test item related trend changes at different intervals. The individual animal variation in all the groups across the time intervals was very high and thus the changes were inconsistent and could not be related to test item administration.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The test item-related microscopic changes were noted in small intestinal segments mainly in jejunum and also in duodenum and ileum, liver, adrenals, stomach, spleen and thymus at different time intervals.
Findings in Repeat dose group:
Adrenal: cortical cell hypertrophy involving all three layers of adrenal (zona glomerulosa, zona fasciculata and zona reticularis) was noted in all treated group males and females.
Duodenum: Lymphangiectasis in 600 mg/kg males and females.
Jejunum: Lymphangiectasis noted in all treated group males and females.
Ileum with Peyer’s patch: Lymphangiectasis in 100 and 600 mg/kg males and in ≥300 mg/kg females.
Liver: cytoplasmic rarefaction/vacuolation in hepatocytes of all treated males and females, increased sinusoidal cells in males at 600 mg/kg and in ≥300 mg/kg females.
Spleen: Increased extra medullary hematopoiesis and lymphoid depletion at 600 mg/kg in males and females.
Stomach: Non glandular epithelial hyperplasia in 600 mg/kg males and 300 mg/kg females, non-glandular ulcer in 600 mg/kg males and females and glandular stomach ulcer in 600 mg/kg males.
Thymus: Lymphoid depletion in ≥ 300 mg/kg males and in all treated group females.

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

Number of post implantation loss was increased at high dose group

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

Two of six dams of the high dose group had complete resorptions.
Dams with any resorption were seen in 0, 100, 300 and 600 mg/kg/day group (i.e.1, 3, 3, 2, respectively)

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

incresase in early and late resorption observed at 600 mg/kg/day dams

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

on fetus was found dead in the high dose group

Other effects:

effects observed, treatment-related

Description (incidence and severity):

In the EFD phase rats at the highest dose 600 mg/kg there was significant reduction in maternal body weights gains during later phase of pregnancy (post GD 11) due to fetal resorptions and with reduced food intake due to maternal stress.
The caesarean parameters at 600 mg/kg, the gravid uterine weights were lower (-49%), with increase in post implantation loss and late resorptions and with 2 incidences of complete resorptions.
There was one incidence of dead fetus and mean weight of male and female fetuses were marginally lower (5 to 9%) at 600 mg/kg dose.
Fetal external and visceral observations did not reveal any abnormalities

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

early or late resorptions

total litter losses by resorption

Description (incidence and severity):

There was one incidence of dead fetus and mean weight of male and female fetuses were marginally lower (5 to 9%) at 600 mg/kg dose.
Fetal external and visceral observations did not reveal any abnormalities

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

changes in litter size and weights

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to maternal toxicity:

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

Dose response relationship:

yes

Relevant for humans:

no

Conclusions:

The evaluated “No Observed Adverse Effect Level (NOAEL)” for Maternal toxicity and Fetal developmental toxicity is 300 mg/kg/day in Wistar rats when Hostanox O 3 P was administered orally by gavage during GD 5 to 19 under the test conditions and doses employed in this study.

Executive summary:

With reference to the effects observed in several repeated dose studies and the developmental toxicity study (low body weight and body weight gain, histopathological changes in adrenals and thymus and resorptions) a 14 day short term oral toxicity study with special focus on the reported effects was performed in rats (non-pregnant and pregnant). This study followed in principle the design as lined out in OECD 407 and 414 (referred as “mechanistic study”). The test item, Hostanox O 3 P, was administered to Wistar rats orally (by gavage) at the doses of 100 (low dose), 300 (Mid dose) and 600 mg/kg/day (to 3 cohorts of rats: the repeated dose phase (14 days), the satellite group (3 and 10 days treatment) and the Embryo Fetal Developmental phase (treatment from gestation day 5 to 19). The repeated dose phase and the satellite group consisted of 6 rats/sex/group, while the Embryo Fetal Developmental phase consisted of 6 presumed pregnant rats (gestation day 0).
In this study, assessments included clinical signs, body weights, food consumption, clinical laboratory investigations of blood (for hematology, acute phase proteins, clinical chemistry and hormone analysis), gross pathology, and histopathological evaluation (repeat dose phase and satellite group). The microscopic examination was initially performed for the control and high dose groups of males and females, and extended to the following organs of lower dose groups as higher incidence of lesions was present in high dose groups. For the Embryo Fetal Developmental phase rats, caesarean section was performed for all rats on GD 20 and dams were examined for gross pathological changes. The uterus from all the animals were removed (by laparotomy) and the contents were examined. The uteri were weighed and examined for the number of implantation sites, early and late resorptions, and number of live and dead fetuses. The number of corpora lutea was counted on each ovary. All the fetuses were sexed, weighed and examined for external malformations. Approximately half the number of fetuses from each dam was examined for visceral malformations.

The following observations were reported:

1.     Immunological response
- increased number of neutrophils, leukocytes and monocytes from day 10 on at 300 and 600 mg/kg bw/d (highest dose tested)
-increase in inflammatory markers at day 10-group in all animals at doses≥ 300 mg/kg bw/d, at day 14-group at 600 mg/kg bw/d dose group and for the developmental phase rats (Gestation Day 20) at all dose groups.

- histopathological changes of thymus and spleen at 600 mg/kg bw/d and
-lymphangiectasis in the gastro intestinal tract from day 3 onwards≥ 300 mg/kg bw/d

2.    Adrenals

Histopathological changes were reported from day 3 onwards. However, it has to be emphasized that the hormonal status remains unchanged and comparable to that of the control animals. Therefore, an endocrine activity is excluded.

3.     Liver
- the bilirubin concentration in blood is increased at 300 and 600 mg/kg bw/d.
- furthermore, the AST and ALT level as well as the triglyceride level are slightly elevated at 600 mg/kg bw/d
- vacuolization of hepatocytes from day 3 on at 300 and 600 mg/kg bw/d.

4.     Stomach
- hyperplasia and ulcers in non-glandular and glandular stomach.

5.     Litter data
- increase in post implantation loss and late resorptions at 600 mg/kg bw/d which is considered to be a secondary effect triggered by the above described histological changes which were already observed from days 3 or 10 onwards in the mid and high dose animals
- at 600 mg/kg/day, there was an incidence of dead fetus and the mean weight of male and female fetuses were marginally lower (5 to 9%)

Fetal external and visceral observations did not reveal any abnormalities.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

reliable without restrictions

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

The feeding of DTB-glycolester at dietary levels of 0, 0.16, 0.4 or 1% (corresponding to 0, 70, 200 and 500 mg/kg bw/d considering a diet conversion factor (ppm to mg/kg bw/d) of 20) to rats resulted in slight growth depression and relatively low food intake in male parent rats of the mid and high dose group. The only change observed in the litters consisted of slight growth retardation of the pups in the top dose group during the final stage of the lactation period. The other data on fertility, lactation performance and survival did not reveal any abnormalities.

From the present reproduction study it is therefore concluded that the feeding of the test substance at dietary levels up to 1.0% failed to induce any deleterious effects, other than slight growth depression in parent males at 0.4 and 1.0% and in pups at 1.0%. The F1 -generation was used for a subsequent chronic toxicity study (130 weeks duration, please refer to IUCLID Chapter 7.5.1). No visceral malformations could be found at the termination of this chronic study and therefore, no teratogenic effects were recorded. Furthermore, the rate of mortality was not affected, the body weight differences of rats of the top-dose group with the control animals were smaller than 10% and there were no outstanding differences in food intake observed during the chronic toxicity study performed in the F1 -generation.
The NOAEL (maternal and developmental) was considered to be 1% (corresponding to 500 mg/kg bw/d; highest dose tested) in diet. None of the fertility/viability parameters examined was adversely affected by the feeding of the test substance. Slightly reduced body weight was observed in males and pups of the high dose group.

In a second study the feeding of DTB glycolester at dietary levels of 0 (control), 0.1, 0.4 or 1.6% to rats for one generation resulted in slight growth depression of the male parent animals at the mid- and high dose level, and decreased body weights and increased mortality of the pups during lactation, at the high dose level only. The growth depression of the male parent rats was accompanied by relatively low food intake and food efficiency figures. The lower food intake may be ascribed to unpalatability of the test diets, but the lower food efficiencies suggest that the growth depression is attributed at least partly to toxic properties of the test substance. The marked growth depression of the offspring in the high dose group during lactation at post-natal day 10 and 20 was accompanied by mortality in this group. The finding of decreased body weight of the pups starting already at day 10 suggests, that this effect does not result directly from ingestion of the test substance with the diet, but rather from change induced already during intra uterine life. Taking into account the log Pow of 7 and the high lipophilic properties of the test substance an increased uptake of DTB glycolester via milk during the lactation phase is also thinkable. The F1 -generation was used for a subsequent chronic toxicity study (16 weeks duration, please refer to IUCLID Chapter 7.5.1). No further mortality during the course of this study or visceral malformations could be found at the termination of this sub-chronic study and therefore, no teratogenic effects were recorded. None of the fertility parameters examined in the one generation study was adversely affected by the feeding of the test substance. A reduced viability and growth depression of the offspring was observed in the high dose group. Due to the restricted reporting the relevance of the observed effects cannot be evaluated and a definite NOAEL cannot be derived.

To investigate if the developmental toxicity observed in the older reproduction study is caused by intrauterine exposure of the conceptus during gestation and/or post-natal exposure of the pups via maternal milk, two Satellite groups (group 5 and 6, consisting of 5 dams each) were included in an OECD 421 guideline study. The rats were fed diets containing 0, 3750, 7500 and 11250 ppm active ingredient (corresponding to nominal dose levels of 0, 250, 500 and 750 mg/kg bw/d, respectively). The additional satellite groups used for cross-fostering were treated in the same manner as Groups 1 to 4 with diets of 0 and11250 ppm. Group 5 was administered the high dose, and Group 6 served as control receiving the standard powder rodent diet without test item. After littering (PND 1), litters of high dose Group 5 were cross-fostered with litters of control Group 6, and vice versa. On PND 11 or 12, from dams used for cross-fostering, blood and milk samples were taken for exposure control.

Signs of toxicity were noted at 11250 ppm and 7500 ppm in parental generation (P0). These consisted of reduced body weight/body weight gain (both sexes), reduced food consumption (females only), and gross findings and microscopic alterations recorded in the adrenal glands (up to marked degree of hemorrhage/congestion and diffuse cystic degeneration of the zona fasciculata, correlating to enlargement and/or discoloration) and thymus (lymphoid depletion at marked degree, correlating to reduced size) in females.

In lactating females at 11250 ppm, reduced body weight gain (7% lower compared to controls) was noted on post-natal day (PND) 13 and 21 resulting in body weights that were respectively 12% and 11% lower compared to controls in the same period. A similar trend towards decreased body weight and body weight gain was observed for lactating females of the 7500 ppm group (mid dose), although not statistically significant.

Lower absolute and relative food consumption was observed in all treated groups during post-coitum and lactation (not always statistically significant) with changes compared to the concurrent control group most pronounced in the high dose group. During the last week of the lactation period (PND 13- 21), mean relative food consumption for females at 11250 ppm and 7500 ppm was 40% and 18%, respectively, lower than for the concurrent control group, while recovery to normal values was seen at the lowest dose level of 3750 ppm.

Test item intake (mean of means) over study period (pre-mating, post-coitum) were determined for males to be 271, 513 and 809 mg test item/kg body weight at dose levels of 3750, 7500, 11250 ppm. For females mean of means of test item intake over study period (pre-mating, post-coitum, lactation) were determined to be 501, 1013 and 1320 mg test item/kg body weight at dose levels of 3750, 7500, 11250 ppm. Bioanalysis for exposure control in plasma during lactation (days 11 or 12) in all high dose females used for cross-fostering revealed that these females were exposed to the test substance in considerable high doses (ranging from 1650 to 5660 ng/mL). Comparable concentrations were also measured in milk of these dams (ranging from 1280 to 5990 ng/mL).

No treatment-related changes were noted in any of the remaining parameters investigated in this study (i.e. viability, clinical appearance, T4 thyroid hormone levels, and organ weights).

No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, pre-coital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

The total number of offspring born compared to the total number of uterine implantations was not considered to be affected by treatment. Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 96, 91, 91 and 95% for the control, 3750, 7500 and 11250 ppm groups, respectively.

The weight at birth of all pups at all dose levels were in the same range as the concurrent control group. Viability of offspring was not affected by treatment with test substance. Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was 99, 100, 99 and 98% for the control, 3750, 7500 and 11250 ppm groups, respectively. For cross-fostered pups the Day 4 viability index was 100% for both the control and 11250 ppm group.

The number of live offspring on Day 21 after littering compared to the number of live offspring on Day 4 (after culling) was not considered to be affected by treatment. One pup in the 3750 ppm was found dead on lactation Day 16, resulting in a lactation index of 98% for this low dose group compared to 100% for all remaining groups. The lactation index for pups after cross-fostering was 100% for both the control and 11250 ppm group. 

A trend towards lower body weight gains was noted in male and female pups of the 11250 ppm and 7500 ppm groups from respectively PND 4 and PND 13 onwards, reaching statistical significance on PND 13 and PND 21 for pups at 11250 ppm group (relative difference to concurrent control group: -19% and -34% for sexes combined) and on PND 21 for pups at 7500 ppm group (relative difference to concurrent control group: -16 % for sexes combined). 

Birth weight of all pups at all dose levels were in the same range as the concurrent control group, indicating that developmental toxicity observed in this study was caused post-natal. This is confirmed by the finding that pups from five control females (i.e. females that had not been administered test item), cross-fostered on PND 1 to five females of 11250 ppm group showed a comparable effect of reduced body weight gain during lactation as pups from high dose dams not cross-fostered , while pups from 11250 ppm females, cross-fostered on PND 1 to control females had normal body weights/body weight gain. Thus, it is assumed, that pups cross-fostered to high dose females were exposed to the test item by lactational transfer in breast milk. This was confirmed by the results of bioanalysis in plasma and milk samples derived from the dams used for cross-fostering. Based on this observation, it is assumed that effects on body weight gain seen in pups from dams treated with 11250 ppm are caused by lactational transfer of test item in breast milk and not intrauterine.

No treatment-related changes were noted in any of the remaining parameters for F1-generation investigated in this study (i.e. viability, clinical appearance, sex-ratio, anogenital distance, areola/nipple retention, T4 thyroid hormone levels). No macroscopic findings were noted among pups, that were considered to be related to treatment.

Toxicity to reproduction: other studies

Description of key information

With reference to the effects observed in several repeated dose studies and the developmental toxicity study (i.e. low body weight and body weight gain, histopathological changes in adrenals and thymus and resorptions) a 14 day short term oral toxicity study with special focus on the reported effects was performed in rats (non-pregnant and pregnant). This study followed in principle the design as lined out in OECD 407 and 414 (referred as “mechanistic study”). The test item, Hostanox O 3 P, was administered to Wistar rats orally (by gavage) at the doses of 100 (low dose), 300 (Mid dose) and 600 mg/kg/day (to 3 cohorts of rats: the repeated dose phase (14 days), the satellite group (3 and 10 days treatment) and the Embryo Fetal Developmental phase (treatment from gestation day 5 to 19). The repeated dose phase and the satellite group consisted of 6 rats/sex/group, while the Embryo Fetal Developmental phase consisted of 6 presumed pregnant rats (gestation day 0).
In this study, assessments included clinical signs, body weights, food consumption, clinical laboratory investigations of blood (for hematology, acute phase proteins, clinical chemistry and hormone analysis), gross pathology, and histopathological evaluation (repeat dose phase and satellite group). The microscopic examination was initially performed for the control and high dose groups of males and females, and extended to the following organs of lower dose groups as higher incidence of lesions was present in high dose groups. For the Embryo Fetal Developmental phase rats, caesarean section was performed for all rats on GD 20 and dams were examined for gross pathological changes. The uterus from all the animals were removed (by laparotomy) and the contents were examined. The uteri were weighed and examined for the number of implantation sites, early and late resorptions, and number of live and dead fetuses. The number of corpora lutea was counted on each ovary. All the fetuses were sexed, weighed and examined for external malformations. Approximately half the number of fetuses from each dam was examined for visceral malformations.

The following observations were reported:

1.     Immunological response:
- increased number of neutrophils, leukocytes and monocytes from day 10 on at 300 and 600 mg/kg bw/d (highest dose tested)
- increase in inflammatory markers at day 10-group in all animals at doses ≥ 300 mg/kg bw/d, at day 14-group at 600 mg/kg bw/d dose group and for the developmental phase rats (Gestation Day 20) at all dose groups.

- histopathological changes of thymus and spleen at 600 mg/kg bw/d and
- lymphangiectasis in the gastro intestinal tract from day 3 onwards ≥ 300 mg/kg bw/d

2.     Adrenals:

Histopathological changes were found from day 3 onwards. However, it has to be emphasized that the hormonal status remains unchanged and comparable to that of the control animals. Therefore, an endocrine activity is excluded.

3.     Liver:
- the bilirubin concentration in blood is increased at 300 and 600 mg/kg bw/d.
- furthermore, the AST and ALT level as well as the triglyceride level are elevated at 600 mg/kg bw/d
- vacuolization of hepatocytes from day 3 on at 300 and 600 mg/kg bw/d.

4.     Stomach
- hyperplasia and ulcers in non-glandular and glandular stomach.

5.     Litter data
- increase in post implantation loss and late resorptions at 600 mg/kg bw/d which is considered to be a secondary effect triggered by the above described histological changes which were already observed from days 3 or 10 onwards in the mid and high dose animals
- at 600 mg/kg/day, there was an incidence of dead fetus and the mean weight of male and female fetuses were marginally lower (5 to 9%)

Fetal external and visceral observations did not reveal any abnormalities.

Based on the above mentioned effects the NOAEL (developmental) was set at 300 mg/kg bw/d.

Justification for classification or non-classification

Based on all available data from four existing studies on reproduction and developmental toxicity with Bis3,3 -Di-(4 -oxy-3 -tert.butyl-phenyl)-butanic acid)-glycol ester, there is no evidence to suggest that a classification for reproductive toxicity is appropriate.

With reference

- to the result reported from a valid reproductive toxicity study combined with a following OECD 451/452 equivalent study performed with the F1-generation resulting in the test design of a one-generation study and

- the result of a valid OECD 421 study including cross-fostering and milk and plamsa analysis of dams, and

- the conclusion that the resorptions observed in the OECD 414 study as well as in the “mechanistic study” are considered as secondary effects caused by primary target organ toxicity,

it is concluded that Bis-(3,3 -Di-(4 -oxy-3 -tert. butyl-phenyl)-butanic acid)-glycol ester is not subject to classification and labelling according to Regulation 1272/2008/EC regarding reproductive toxicity.